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Sample records for 3a4 enzyme involvement

  1. Interactions of ingested food, beverage, and tobacco components involving human cytochrome P4501A2, 2A6, 2E1, and 3A4 enzymes.

    Guengerich, F P; Shimada, T; Yun, C H; Yamazaki, H; Raney, K D; Thier, R; Coles, B; Harris, T M

    1994-01-01

    Human cytochrome P450 (P450) enzymes are involved in the oxidation of natural products found in foods, beverages, and tobacco products and their catalytic activities can also be modulated by components of the materials. The microsomal activation of aflatoxin B1 to the exo-8,9-epoxide is stimulated by flavone and 7,8-benzoflavone, and attenuated by the flavonoid naringenin, a major component of grapefruit. P4502E1 has been demonstrated to play a potentially major role in the activation of a number of very low-molecular weight cancer suspects, including ethyl carbamate (urethan), which is present in alcoholic beverages and particularly stone brandies. The enzyme (P4502E1) is also known to be inducible by ethanol. Tobacco contains a large number of potential carcinogens. In human liver microsomes a significant role for P4501A2 can be demonstrated in the activation of cigarette smoke condensate. Some of the genotoxicity may be due to arylamines. P4501A2 is also inhibited by components of crude cigarette smoke condensate. The tobacco-specific nitrosamines are activated by a number of P450 enzymes. Of those known to be present in human liver, P4501A2, 2A6, and 2E1 can activate these nitrosamines to genotoxic products. PMID:7698084

  2. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    Petersen, Maria Skaalum; Halling, Jónrit; Damkier, Per;

    2007-01-01

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1......,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3......A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6beta-hydroxycortisol/cortisol (6beta...

  3. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6β-hydroxycortisol/cortisol (6β-OHC/FC) ratio. POP exposures were assessed by measuring their concentrations in serum lipid. The results showed a unimodal distribution of the 6β-OHC/FC ratio with values ranging from 0.58 to 27.38. Women had a slightly higher 6β-OHC/FC ratio than men (p = 0.07). Confounder-adjusted multiple regression analysis showed significant associations between 6β-OHC/FC ratios and ΣPCB, PCB-TEQ and p,p'-DDE, o,p'-DDT and HCB, respectively, but the associations were statistically significant for men only

  4. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-02-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  5. 肝药酶CYP3A4与抗癫(癎)药物的代谢研究进展%Progress on the metabolism of antiepileptic drugs by human hepatic CYP3A4 enzymes

    张君梅

    2011-01-01

    癫(癎)是危害人类健康的常见神经系统疾病,临床常用抗癫(癎)药多通过细胞色素P450等肝药酶代谢.CYP3A4是P450酶系中最重要的代谢酶,参与40%~60%的药物代谢,与临床常用抗癫(癎)药的代谢也有非常紧密的关系.该文对CYP3A4的一般特性及其与抗癫(癎)药物的代谢研究进展作一概述.%Epilepsy is a common disease in nervous system.Most antiepileptic drugs are metabolized by liver enzymes.such as cytochrome P450 enzyme.CYP3A4 iS the most important enzyme in P450 family.involved in the metabolism of about 40%~60% of the drugs use for clinic.It also has a close relationship with the metabolism of antiepileptie drugs.This review summarizes the general characteristic of CYP3A4 and itsrelationship with the metabolism of antiepileptic drugs.

  6. High frequency and founder effect of the CYP3A4*20 loss-of-function allele in the Spanish population classifies CYP3A4 as a polymorphic enzyme.

    Apellániz-Ruiz, M; Inglada-Pérez, L; Naranjo, M E G; Sánchez, L; Mancikova, V; Currás-Freixes, M; de Cubas, A A; Comino-Méndez, I; Triki, S; Rebai, A; Rasool, M; Moya, G; Grazina, M; Opocher, G; Cascón, A; Taboada-Echalar, P; Ingelman-Sundberg, M; Carracedo, A; Robledo, M; Llerena, A; Rodríguez-Antona, C

    2015-06-01

    Cytochrome P450 3A4 (CYP3A4) is a key drug-metabolizing enzyme. Loss-of-function variants have been reported as rare events, and the first demonstration of a CYP3A4 protein lacking functional activity is caused by CYP3A4*20 allele. Here we characterized the world distribution and origin of CYP3A4*20 mutation. CYP3A4*20 was determined in more than 4000 individuals representing different populations, and haplotype analysis was performed using CYP3A polymorphisms and microsatellite markers. CYP3A4*20 allele was present in 1.2% of the Spanish population (up to 3.8% in specific regions), and all CYP3A4*20 carriers had a common haplotype. This is compatible with a Spanish founder effect and classifies CYP3A4 as a polymorphic enzyme. This constitutes the first description of a CYP3A4 loss-of-function variant with high frequency in a population. CYP3A4*20 results together with the key role of CYP3A4 in drug metabolism support screening for rare CYP3A4 functional alleles among subjects with adverse drug events in certain populations. PMID:25348618

  7. Gene-gene-environment interactions between drugs, transporters, receptors, and metabolizing enzymes: Statins, SLCO1B1, and CYP3A4 as an example.

    Sadee, Wolfgang

    2013-09-01

    Pharmacogenetic biomarker tests include mostly specific single gene-drug pairs, capable of accounting for a portion of interindividual variability in drug response and toxicity. However, multiple genes are likely to contribute, either acting independently or epistatically, with the CYP2C9-VKORC1-warfarin test panel, an example of a clinically used gene-gene-dug interaction. I discuss here further instances of gene-gene-drug interactions, including a proposed dynamic effect on statin therapy by genetic variants in both a transporter (SLCO1B1) and a metabolizing enzyme (CYP3A4) in liver cells, the main target site where statins block cholesterol synthesis. These examples set a conceptual framework for developing diagnostic panels involving multiple gene-drug combinations. PMID:23436703

  8. Systemic uptake of miconazole during vaginal suppository use and effect on CYP1A2 and CYP3A4 associated enzyme activities in women

    Kjærstad, Mia Birkhøj; Nielsen, Flemming; Nøhr-Jensen, Lene;

    2010-01-01

    To investigate if the ordinary use of a vaginal suppository containing miconazole results in systemic absorption that is sufficient to affect the activities of CYP1A2 and CYP3A4, which are major drug- and steroid-metabolising enzymes....

  9. Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

    Savary, Camille C; Jossé, Rozenn; Bruyère, Arnaud; Guillet, Fabrice; Robin, Marie-Anne; Guillouzo, André

    2014-08-01

    Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation. PMID:24832206

  10. Pyrethroid insecticides: Isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

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

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

    1995-01-01

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

  12. CYP3A4*1G gene Polymorphism on Javanese People

    Em Sutrisna

    2015-11-01

    Full Text Available AbtractMost of drugs are metabolized by cytochrome P 450 (CYP enzyme. Cytochrome P450 3A4 is the cytochrome that is involved in metabolizing more than 60% of all medicine used in human. The variation of this CYP3A4 gene will affect the catalytic activity of this enzyme. Recently, CYP3A4*1G in intron 10 was found in Chinese and Japanese population. There is a substitution of G to A at position 82266 in intron 10. The purpose of this research was to investigate the frequency of allele and genotype CYP3A4*1G. Samples were taken from bloods of the subjects of the research. The examination of CYP3A4*1G was conducted by RTLP-PCR method.As the results of this research, the frequency of CYP3A4*1G in Javanese people is CYP3A4*1/*1 0.25, CYP3A4*1/*1G 0.55 and CYP3A4*1G/*1G 0.20. Frequency of allele G: 0.53, allele A: 0.47. The Fisher’s exact- test shows that the allele and genotype frequencyis p. 1.000. The allele and genotype frequency of Javanese people isstill in Hardy-Weinberg equilibrium.Keywords : CYP3A4*1G gene, polymorphism, Javanese people

  13. Role of Enzyme Flexibility in Ligand Access and Egress to Active Site: Bias-Exchange Metadynamics Study of 1,3,7-Trimethyluric Acid in Cytochrome P450 3A4.

    Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Laio, Alessandro; Otyepka, Michal

    2016-04-12

    Although the majority of enzymes have buried active sites, very little is known about the energetics and mechanisms associated with substrate and product channeling in and out. Gaining direct information about these processes is a challenging task both for experimental and theoretical techniques. Here, we present a methodology that enables following of a ligand during its passage to the active site of cytochrome P450 (CYP) 3A4 and mapping of the free energy associated with this process. The technique is based on a combination of a bioinformatics tool for identifying access channels and bias-exchange metadynamics and provides converged free energies in good agreement with experimental data. In addition, it identifies the energetically preferred escape routes, limiting steps, and amino acids residues lining the channel. The approach was applied to mapping of a complex channel network in a complex environment, i.e., CYP3A4 attached to a lipid bilayer mimicking an endoplasmic reticulum membrane. The results provided direct information about the energetics and conformational changes associated with the ligand channeling. The methodology can easily be adapted to study channeling through other flexible biomacromolecular channels. PMID:26967371

  14. High-throughput fluorescence assay of cytochrome P450 3A4

    Cheng, Qian; Guengerich, F. Peter

    2013-01-01

    Microtiter plate-based fluorescence assays allow rapid measurement of the catalytic activities of cytochrome P450 oxygenases (P450s). We describe a high-throughput fluorescence assay of P450 3A4, one of the key enzymes involved in xenobiotic metabolism. The assay involves the oxidative debenzylation of 7-hydroxy-4-trifluoromethyl coumarin, producing an increase in fluorescence.

  15. Enzymes involved in organellar DNA replication in photosynthetic eukaryotes

    Takashi eMoriyama

    2014-09-01

    Full Text Available Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

  16. Lupine protein hydrolysates inhibit enzymes involved in the inflammatory pathway

    Millán-Linares, María del Carmen; Yust, María del Mar; Alcaide-Hidalgo, J. M.; Millán, Francisco; Pedroche, Justo

    2014-01-01

    Lupine protein hydrolysates (LPHs) were obtained from a lupine protein isolate (LPI) by enzymatic hydrolysis using two proteases, Izyme AL and Alcalase 2.4 L, and their potential anti-inflammatory capacities were studied by determining their in vitro inhibition of the following enzymes that are involved in the inflammatory process: phospholipase A2 (PLA2), cyclooxygenase 2 (COX-2), thrombin, and transglutaminase (TG). The strongest inhibitory activities toward PLA2 and TG were found in the hy...

  17. The effect of interferon-α on the expression of cytochrome P450 3A4 in human hepatoma cells

    Interferon α (IFNα) is used to treat malignancies and chronic viral infections. It has been found to decrease the rate of drug metabolism by acting on cytochrome P450 enzymes, but no studies have investigated the consequences of IFNα treatment on the CYP3A4 isoform, responsible for the metabolism of a majority of drugs. In this study, we have examined the effect of IFNα on CYP3A4 catalytic activity and expression in human hepatoma cells. We found that IFNα inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4, independent of de novo protein synthesis. Pharmacologic inhibitors and a dominant-negative mutant expression plasmid were used to dissect the molecular pathway required for CYP3A4 suppression, revealing roles for Jak1 and Stat1 and eliminating the involvement of the p38 mitogen-activated and extracellular regulated kinases. Treatment of hepatoma cells with IFNα did not affect the nuclear localization or relative abundance of Sp1 and Sp3 transcription factors, suggesting that the suppression of CYP3A4 by IFNα does not result from inhibitory Sp3 out-competing Sp1. To our knowledge, this is the first report that IFNα down-regulates CYP3A4 expression largely through the JAK-STAT pathway. Since IFNα suppresses CYP3A4 expression, caution is warranted when IFNα is administered in combination with CYP3A4 substrates to avoid the occurrence of adverse drug interactions.

  18. Lipolytic Enzymes Involved in the Virulence of Human Pathogenic Fungi

    Park, Minji; Do, Eunsoo; Jung, Won Hee

    2013-01-01

    Pathogenic microbes secrete various enzymes with lipolytic activities to facilitate their survival within the host. Lipolytic enzymes include extracellular lipases and phospholipases, and several lines of evidence have suggested that these enzymes contribute to the virulence of pathogenic fungi. Candida albicans and Cryptococcus neoformans are the most commonly isolated human fungal pathogens, and several biochemical and molecular approaches have identified their extracellular lipolytic enzym...

  19. Accessory enzymes from Aspergillus involved in xylan and pectin degradation

    Vries, de, G.

    1999-01-01

    The xylanolytic and pectinolytic enzyme systems from Aspergillus have been the subject of study for many years. Although the main chain cleaving enzymes and their encoding genes have been studied in detail, little information is available about most of the accessory enzymes and their corresponding genes. This thesis describes the purification and characterisation of two accessory enzymes from Aspergillus , feruloyl esterase A (FaeA) andα-glucuronidase A (AguA), and the activities of these enz...

  20. Enzymes involved in organellar DNA replication in photosynthetic eukaryotes

    Moriyama, Takashi; Sato, Naoki

    2014-01-01

    Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana...

  1. Identification of human cytochrome P450 enzymes involved in the hepatic and intestinal biotransformation of 20(S)-protopanaxadiol.

    Chiu, Nga Ting Colette; Tomlinson Guns, Emma S; Adomat, Hans; Jia, William; Deb, Subrata

    2014-03-01

    20(S)-Protopanaxadiol (aPPD), a ginseng sapogenin, has been shown to be a promising anti-cancer compound and anti-depressant agent. Although the bacterial biotransformation of ginsenosides has been studied thoroughly, few have reported on the cytochrome P450 (P450) mediated metabolism of aPPD. Taken orally, aPPD must first undergo absorption and metabolism in the intestine before further metabolism in the liver. The present study investigated the comparative biotransformation profile of aPPD in human intestinal microsomes (HIM) and human liver microsomes (HLM) and characterized the human P450 enzymes involved in aPPD metabolism. Three major monooxygenated metabolites and five minor dioxygenated metabolites were identified as the predominant products in aPPD incubations with HIM and HLM using liquid chromatography-mass spectrometry. Reaction phenotyping studies were performed with a panel of specific P450 chemical inhibitors, antibody inhibition and human recombinant P450 enzymes. Ketoconazole, a CYP3A inhibitor, blocked the formation of oxygenated metabolites of aPPD in both HIM and HLM in a concentration dependent manner. Among the human recombinant P450 enzymes assayed, CYP3A4 exhibited the highest activity towards aPPD oxidative metabolite formation, followed by CYP3A5. In summary, the results have shown that aPPD is extensively metabolized by HIM and the metabolite profile following in vitro incubations is similar in HIM and HLM. CYP3A4 and CYP3A5 isoforms are the predominant enzymes responsible for oxygenation of aPPD in HIM and HLM. The characterization of aPPD as a CYP3A substrate may facilitate better prediction of drug-herb interactions when aPPD is taken concomitantly with other therapeutic agents. PMID:24151189

  2. Microbial urea-formaldehyde degradation involves a new enzyme, methylenediurease.

    Jahns, T; Schepp, R; Siersdorfer, C; Kaltwasser, H

    1998-01-01

    The enzymic mechanism of metabolization of urea-formaldehyde condensation products (methyleneureas; MU) and the fate of the degradation products ammonium, urea and formaldehyde were studied in bacteria isolated from garden soil, which were able to use methyleneureas as the sole source of nitrogen for growth. An organism identified as Ochrobactrum anthropi completely degraded methylenediurea (MDU) and dimethylenetriurea (DMTU) to urea, ammonia, formaldehyde and carbon dioxide. An enzyme designated as methylenediurease (methylenediurea deiminase; MDUase) was responsible for the degradation of both MDU and DMTU as well as higher polymerized MU. Growth on MU as the nitrogen source specifically induced the synthesis of this enzyme, which seems to be located in the periplasm of the bacterium. Under these growth conditions, urease as well as NAD-specific formaldehyde and formiate dehydrogenase were expressed to high levels, efficiently using the products of MU degradation, and high-affinity transport systems for urea and ammonia were synthesized scavenging the environment for these products. PMID:10526991

  3. Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.

    Davydov, Dmitri R; Yang, Zhongyu; Davydova, Nadezhda; Halpert, James R; Hubbell, Wayne L

    2016-04-12

    We used high hydrostatic pressure as a tool for exploring the conformational landscape of human cytochrome P450 3A4 (CYP3A4) by electron paramagnetic resonance and fluorescence spectroscopy. Site-directed incorporation of a luminescence resonance energy transfer donor-acceptor pair allowed us to identify a pressure-dependent equilibrium between two states of the enzyme, where an increase in pressure increased the spatial separation between the two distantly located fluorophores. This transition is characterized by volume change (ΔV°) and P1/2 values of -36.8 ± 5.0 mL/mol and 1.45 ± 0.33 kbar, respectively, which corresponds to a Keq° of 0.13 ± 0.06, so that only 15% of the enzyme adopts the pressure-promoted conformation at ambient pressure. This pressure-promoted displacement of the equilibrium is eliminated by the addition of testosterone, an allosteric activator. Using site-directed spin labeling, we demonstrated that the pressure- and testosterone-sensitive transition is also revealed by pressure-induced changes in the electron paramagnetic resonance spectra of a nitroxide side chain placed at position 85 or 409 of the enzyme. Furthermore, we observed a pressure-induced displacement of the emission maxima of a solvatochromic fluorophore (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin) placed at the same positions, which suggests a relocation to a more polar environment. Taken together, the results reveal an effector-dependent conformational equilibrium between open and closed states of CYP3A4 that involves a pronounced change at the interface between the region of α-helices A/A' and the meander loop of the enzyme, where residues 85 and 409 are located. Our study demonstrates the high potential of pressure-perturbation strategies for studying protein conformational landscapes. PMID:27074675

  4. Amlodipine metabolism in human liver microsomes and roles of CYP3A4/5 in the dihydropyridine dehydrogenation.

    Zhu, Yanlin; Wang, Fen; Li, Quan; Zhu, Mingshe; Du, Alicia; Tang, Wei; Chen, Weiqing

    2014-02-01

    Amlodipine is a commonly prescribed calcium channel blocker for the treatment of hypertension and ischemic heart disease. The drug is slowly cleared in humans primarily via dehydrogenation of its dihydropyridine moiety to a pyridine derivative (M9). Results from clinical drug-drug interaction studies suggest that CYP3A4/5 mediate metabolism of amlodipine. However, attempts to identify a role of CYP3A5 in amlodipine metabolism in humans based on its pharmacokinetic differences between CYP3A5 expressers and nonexpressers failed. Objectives of this study were to determine the metabolite profile of amlodipine (a racemic mixture and S-isomer) in human liver microsomes (HLM), and to identify the cytochrome P450 (P450) enzyme(s) involved in the M9 formation. Liquid chromatography/mass spectrometry analysis showed that amlodipine was mainly converted to M9 in HLM incubation. M9 underwent further O-demethylation, O-dealkylation, and oxidative deamination to various pyridine derivatives. This observation is consistent with amlodipine metabolism in humans. Incubations of amlodipine with HLM in the presence of selective P450 inhibitors showed that both ketoconazole (an inhibitor of CYP3A4/5) and CYP3cide (an inhibitor of CYP3A4) completely blocked the M9 formation, whereas chemical inhibitors of other P450 enzymes had little effect. Furthermore, metabolism of amlodipine in expressed human P450 enzymes showed that only CYP3A4 had significant activity in amlodipine dehydrogenation. Metabolite profiles and P450 reaction phenotyping data of a racemic mixture and S-isomer of amlodipine were very similar. The results from this study suggest that CYP3A4, rather than CYP3A5, plays a key role in metabolic clearance of amlodipine in humans. PMID:24301608

  5. Identification of a functional homolog of the mammalian CYP3A4 in locusts

    Olsen, Line Rørbæk; Gabel-Jensen, Charlotte; Nielsen, Peter Aadal; Hansen, Steen Honoré; Badolo, Lassina

    2014-01-01

    mammalian CYP3A4 inhibitor, suggesting that the enzyme responsible for the human metabolite formation in locusts is functionally similar to human CYP3A4. Besides the human metabolites of terfenadine, additional metabolites were formed in locusts. These were tentatively identified as phosphate and glucose...

  6. SEARCH FOR ENZYMES INVOLVED IN THE TRINITROTOLUENE DEGRADATION IN PLANTS

    Podlipná, Radka; Vavříková, Zuzana; Vágner, Martin; Vaněk, Tomáš

    University of Verona, 2008. s. 29-30. [Genes and Proteins Involved in Steps of Phytoextraction and Degradation of Pollutants. 05.06.2008-06.06.2008, Verona] Institutional research plan: CEZ:AV0Z50380511 Keywords : Trinitrotoluene * Saponaria officinalis * nitroreductases Subject RIV: DJ - Water Pollution ; Quality

  7. A RALDH-like enzyme involved in Fusarium verticillioides development

    Díaz-Sánchez, Violeta

    2015-12-11

    Retinaldehyde dehydrogenases (RALDHs) convert retinal to retinoic acid, an important chordate morphogen. Retinal also occurs in some fungi, such as Fusarium and Ustilago spp., evidenced by the presence of rhodopsins and β–carotene cleaving, retinal-forming dioxygenases. Based on the assumption that retinoic acid may also be formed in fungi, we searched the Fusarium protein databases for RALDHs homologs, focusing on Fusarium verticillioides. Using crude lysates of Escherichia coli cells expressing the corresponding cDNAs, we checked the capability of best matches to convert retinal into retinoic acid in vitro. Thereby, we identified an aldehyde dehydrogenase, termed CarY, as a retinoic acid-forming enzyme, an activity that was also exerted by purified CarY. Targeted mutation of the carY gene in F. verticillioides resulted in alterations of mycelia development and conidia morphology in agar cultures, and reduced capacity to produce perithecia as a female in sexual crosses. Complementation of the mutant with a wild-type carY allele demonstrated that these alterations are caused by the lack of CarY. However, retinoic acid could not be detected by LC-MS analysis either in the wild type or the complemented carY strain in vivo, making elusive the connection between CarY enzymatic activity and retinoic acid formation in the fungus.

  8. Phosphorylating enzymes involved in glucose fermentation of Actinomyces naeslundii.

    Takahashi, N.; Kalfas, S; Yamada, T.

    1995-01-01

    Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucose-grown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6-phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PPi. Phos...

  9. Microsomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 2,4-dichlorophenol-An endocrine disruptor compound

    Cytochrome P450-3A4 (CYP3A4) is a monooxygenase enzyme that plays a major role in the detoxification of bioactive compounds and hydrophobic xenobiotics (e.g. medicines, drugs, environmental pollutants, food supplements and steroids). Physiologically the monooxygenation reactions of this class II, microsomal, b-type heme enzyme, usually requires cytochrome P450 reductase, NADPH. A novel CYP3A4 biosensor system that essentially simplified the enzymatic redox processes by allowing electron transfer between the electrode and the enzyme redox centre to occur, without any need for the physiological redox partners, was developed for the detection of 2,4-dichlorophenol (2,4-DCP), a priority environmental pollutant and an endocrine disruptor. The biosensor, GC/Naf-Co(Sep)3+/CYP3A4/Naf, was constructed by encapsulating CYP3A4 in a Nafion-cobalt (III) sepulchrate (Naf-Co(Sep)3+) composite film on a glassy carbon (GC) electrode. The responses of the biosensor to 2,4-dichlorophenol, erythromycin (CYP3A4 native substrate) and ketoconazole (CYP 3A4 natural inhibitor) were studied by cyclic and square wave voltammetric techniques. The detection limit (DL) of the biosensor for 2,4-dichlorophenol was 0.043 μg L-1, which is by an order of magnitude lower than the EU limit (0.3 μg L-1) for any pesticide compound in ground water. The biosensor's DL is lower than the U.S. Environmental Protection Agency's drinking water equivalent level (DWEL) value for 2,4-DCP, which is 2 μg L-1

  10. Microsomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 2,4-dichlorophenol-An endocrine disruptor compound

    Hendricks, Nicolette R.; Waryo, Tesfaye T.; Arotiba, Omotayo; Jahed, Nazeem; Baker, Priscilla G.L. [SensorLab, Department of Chemistry, University of Western Cape, Moderddam Road, Bellville, Cape Town 7535 (South Africa); Iwuoha, Emmanuel I. [SensorLab, Department of Chemistry, University of Western Cape, Moderddam Road, Bellville, Cape Town 7535 (South Africa)], E-mail: eiwuoha@uwc.ac.za

    2009-02-28

    Cytochrome P450-3A4 (CYP3A4) is a monooxygenase enzyme that plays a major role in the detoxification of bioactive compounds and hydrophobic xenobiotics (e.g. medicines, drugs, environmental pollutants, food supplements and steroids). Physiologically the monooxygenation reactions of this class II, microsomal, b-type heme enzyme, usually requires cytochrome P450 reductase, NADPH. A novel CYP3A4 biosensor system that essentially simplified the enzymatic redox processes by allowing electron transfer between the electrode and the enzyme redox centre to occur, without any need for the physiological redox partners, was developed for the detection of 2,4-dichlorophenol (2,4-DCP), a priority environmental pollutant and an endocrine disruptor. The biosensor, GC/Naf-Co(Sep){sup 3+}/CYP3A4/Naf, was constructed by encapsulating CYP3A4 in a Nafion-cobalt (III) sepulchrate (Naf-Co(Sep){sup 3+}) composite film on a glassy carbon (GC) electrode. The responses of the biosensor to 2,4-dichlorophenol, erythromycin (CYP3A4 native substrate) and ketoconazole (CYP 3A4 natural inhibitor) were studied by cyclic and square wave voltammetric techniques. The detection limit (DL) of the biosensor for 2,4-dichlorophenol was 0.043 {mu}g L{sup -1}, which is by an order of magnitude lower than the EU limit (0.3 {mu}g L{sup -1}) for any pesticide compound in ground water. The biosensor's DL is lower than the U.S. Environmental Protection Agency's drinking water equivalent level (DWEL) value for 2,4-DCP, which is 2 {mu}g L{sup -1}.

  11. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A. [Departments of Biochemistry and Chemistry, University of Illinois, 505 South Goodwin Avenue (United States); Sligar, Stephen G., E-mail: s-sligar@illinois.edu [Departments of Biochemistry and Chemistry, University of Illinois, 505 South Goodwin Avenue (United States)

    2013-01-25

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  12. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  13. Quinine metabolism in man : Emphasis on the 3-hydroxylation as a biomarker reaction for the activity of CYP3A4

    Mirghani, Rajaa A

    2002-01-01

    Quinine is one of the cinchona alkaloids and used in the treatment of severe forms of malaria. Formation of 3-hydroxyquinine is catalysed by cytochrome P-450 3A4 (CYP3A4), which is the enzyme involved in the metabolism of about 50% of all drugs in clinical use. In this thesis 1 investigated the relative importance of four metabolites of quinine in its metabolism in vitro and in vivo and studied the usefulness of the metabolic ratio (MR) of quinine (quinine/3- hydroxyquin...

  14. CYP3A4 activity reduces the cytotoxic effects of okadaic acid in HepaRG cells.

    Kittler, Katrin; Fessard, Valérie; Maul, Ronald; Hurtaud-Pessel, Dominique

    2014-08-01

    The biotoxin okadaic acid (OA), produced by dinoflagellates in marine environment, can accumulate in sponges and shellfish. Consumption of contaminated shellfish induces acute toxic effects such as diarrhea, nausea, vomiting, and abdominal pain. CYP3A4, one of the most important human xenobiotic metabolizing enzymes, is supposed to be involved in the metabolism of OA. Aim of our study was to evaluate the role of CYP3A4 in OA in vitro metabolism as well as in cell cytotoxicity in parallel. Therefore, a metabolic competent HepaRG cell line was exposed to OA with and without addition of the CYP3A4 inhibitor ketoconazole. Without the inhibitor, two mono-hydroxylated metabolites could be identified, whereas in its presence, no metabolites could be detected. Confirmation of the formed metabolites was accomplished by measuring the exact masses and investigating the fragmentation pattern. Data obtained from cytotoxicity assays showed that OA cytotoxicity is reduced when CYP3A4 is active. Thus, hydroxylation appears to be a crucial step for metabolic OA detoxification. PMID:24504163

  15. Investigation into CYP3A4-mediated drug–drug interactions on midostaurin in healthy volunteers

    Dutreix, Catherine; Munarini, Florence; Lorenzo, Sebastien; Roesel, Johannes; Wang, Yanfeng

    2013-01-01

    Purpose Midostaurin (PKC412), a multitargeted tyrosine kinase inhibitor that targets FMS-related tyrosine kinase 3 and KIT, is in clinical trials for the treatment for acute myeloid leukemia and advanced systemic mastocytosis. In vitro studies showed that midostaurin is predominantly metabolized by cytochrome P450 3A4 (CYP3A4) and that midostaurin inhibits and/or induces the same enzyme. Here, we address the clinical relevance of CYP3A4-related drug–drug interactions with midostaurin as eithe...

  16. Discovery of a Highly Selective CYP3A4 Inhibitor Suitable for Reaction Phenotyping Studies and Differentiation of CYP3A4 and CYP3A5

    Li, Xiaohai; Song, Xinyi; Kamenecka, Theodore M.; Cameron, Michael D.

    2012-01-01

    Current molecular tools lack the ability to differentiate the activity of CYP3A4 and CYP3A5 in biological samples such as human liver microsomes. Kinetic experiments and the CYP3A4 crystal structure indicate that the active sites of both enzymes are large and flexible, and have more than one binding subsite within the active site. 1-(4-Imidazopyridinyl-7phenyl)-3-(4′-cyanobiphenyl) urea (SR-9186) was optimized through several rounds of structural refinement from an initial screening hit to ob...

  17. Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate

    Dhirendra K Simanshu; Sagar Chittori; H S Savithri; M R N Murthy

    2007-09-01

    In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes.

  18. Enzymes involved in vinyl acetate decomposition by Pseudomonas fluorescens PCM 2123 strain

    Szczyrba, Elżbieta; Greń, Izabela; Bartelmus, Grażyna

    2013-01-01

    Esterases are widely used in food processing industry, but there is little information concerning enzymes involved in decompositions of esters contributing to pollution of environment. Vinyl acetate (an ester of vinyl alcohol and acetic acid) is a representative of volatile organic compounds (VOCs) in decomposition, of which hydrolyses and oxidoreductases are mainly involved. Their activities under periodically changing conditions of environment are essential for the removal of dangerous VOCs...

  19. Sugarcane expressed sequences tags (ESTs encoding enzymes involved in lignin biosynthesis pathways

    Ramos Rose Lucia Braz

    2001-01-01

    Full Text Available Lignins are phenolic polymers found in the secondary wall of plant conductive systems where they play an important role by reducing the permeability of the cell wall to water. Lignins are also responsible for the rigidity of the cell wall and are involved in mechanisms of resistance to pathogens. The metabolic routes and enzymes involved in synthesis of lignins have been largely characterized and representative genes that encode enzymes involved in these processes have been cloned from several plant species. The synthesis of lignins is liked to the general metabolism of the phenylpropanoids in plants, having enzymes (e.g. phenylalanine ammonia-lyase (PAL, cinnamate 4-hydroxylase (C4H and caffeic acid O-methyltransferase (COMT common to other processes as well as specific enzymes such as cinnamoyl-CoA reductase (CCR and cinnamyl alcohol dehydrogenase (CAD. Some maize and sorghum mutants, shown to have defective in CAD and/or COMT activity, are easier to digest because they have a reduced lignin content, something which has motivated different research groups to alter the lignin content and composition of model plants by genetic engineering try to improve, for example, the efficiency of paper pulping and digestibility. In the work reported in this paper, we have made an inventory of the sugarcane expressed sequence tag (EST coding for enzymes involved in lignin metabolism which are present in the sugarcane EST genome project (SUCEST database. Our analysis focused on the key enzymes ferulate-5-hydroxylase (F5H, caffeic acid O-methyltransferase (COMT, caffeoyl CoA O-methyltransferase (CCoAOMT, hydroxycinnamate CoA ligase (4CL, cinnamoyl-CoA reductase (CCR and cinnamyl alcohol dehydrogenase (CAD. The comparative analysis of these genes with those described in other species could be used as molecular markers for breeding as well as for the manipulation of lignin metabolism in sugarcane.

  20. Characterization of Human Hepatic and Extrahepatic UDP-Glucuronosyltransferase Enzymes Involved in the Metabolism of Classic Cannabinoids

    Mazur, Anna; Lichti, Cheryl F.; Prather, Paul L.; Zielinska, Agnieszka K.; Bratton, Stacie M.; Gallus-Zawada, Anna; Finel, Moshe; Miller, Grover P.; Radomińska-Pandya, Anna; Moran, Jeffery H.

    2009-01-01

    Tetrahydrocannabinol (Δ9-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible for these cytochrome P450-dependent oxidations, but little work has been done to characterize phase II metabolic pathways. In this study, we test the hypothesis that there are specific human UG...

  1. A theoretical study of the molecular mechanism of the GAPDH Trypanosoma cruzi enzyme involving iodoacetate inhibitor

    Carneiro, Agnaldo Silva; Lameira, Jerônimo; Alves, Cláudio Nahum

    2011-10-01

    The glyceraldehyde-3-phosphate dehydrogenase enzyme (GAPDH) is an important biological target for the development of new chemotherapeutic agents against Chagas disease. In this Letter, the inhibition mechanism of GAPDH involving iodoacetate (IAA) inhibitor was studied using the hybrid quantum mechanical/molecular mechanical (QM/MM) approach and molecular dynamic simulations. Analysis of the potential energy surface and potential of mean force show that the covalent attachment of IAA inhibitor to the active site of the enzyme occurs as a concerted process. In addition, the energy terms decomposition shows that NAD+ plays an important role in stabilization of the reagents and transition state.

  2. GW4064, an agonist of farnesoid X receptor, represses CYP3A4 expression in human hepatocytes by inducing small heterodimer partner expression.

    Zhang, Shu; Pan, Xian; Jeong, Hyunyoung

    2015-05-01

    Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis and is recognized as a promising therapeutic target for metabolic diseases. The biologic function of FXR is mediated in part by a small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, and SHP in turn represses the activity of multiple transcription factors. This study aimed to investigate the effect of FXR activation on expression of the major drug-metabolizing enzyme CYP3A4. The effects of 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), a synthetic agonist of FXR, on the expression and activity of CYP3A4 were examined in primary human hepatocytes by using quantitative real-time polymerase chain reaction and S9 phenotyping. In human hepatocytes, treatment of GW4064 (1 μM) for 48 hours resulted in a 75% decrease in CYP3A4 mRNA expression and a 25% decrease in CYP3A4 activity, accompanied by ∼3-fold increase in SHP mRNA expression. In HepG2 cells, SHP repressed transactivation of CYP3A4 promoter by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and glucocorticoid receptor. Interestingly, GW4064 did not repress expression of CYP2B6, another target gene of PXR and CAR; GW4064 enhanced CYP2B6 promoter activity. In conclusion, GW4064 represses CYP3A4 expression in human hepatocytes, potentially through upregulation of SHP expression and subsequent repression of CYP3A4 promoter activity. Clinically significant drug-drug interaction involving FXR agonists and CYP3A4 substrates may occur. PMID:25725071

  3. Enzyme

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  4. Studies on enzymes involved in DNA synthesis and thymine nucleotide formation in potato tuber slices

    Activity changes of several enzymes involved in DNA synthesis were investigated in potato tuber tissue in which DNA synthesis was induced by slicing. Nucleoside phosphotransferase activity increased only slightly during aging of the tissue discs. Thymidine monophosphate (TMP) kinase activity increased about 36% after aging for 24 hr. Protein synthesis in an early stage of aging was necessary for the activity increase. A 2.7-fold increase was observed in DNA polymerase activity after aging for 36 hr. The activity increase was due to continuous synthesis of enzyme protein. In vivo examination of TMP synthetase suggests that its activity does not necessarily increase before full development of DNA synthesis. It was concluded that among the enzymes examined, TMP kinase activity may increase shortly after slicing to support a massive supply of thymidine triphosphate and the increased activity of DNA polymerane may contribute to the active synthesis of DNA in aged discs. (auth.)

  5. Ethylbenzene Dehydrogenase and Related Molybdenum Enzymes Involved in Oxygen-Independent Alkyl Chain Hydroxylation.

    Heider, Johann; Szaleniec, Maciej; Sünwoldt, Katharina; Boll, Matthias

    2016-01-01

    Ethylbenzene dehydrogenase initiates the anaerobic bacterial degradation of ethylbenzene and propylbenzene. Although the enzyme is currently only known from a few closely related denitrifying bacterial strains affiliated to the Rhodocyclaceae, it clearly marks a universally occurring mechanism used for attacking recalcitrant substrates in the absence of oxygen. Ethylbenzene dehydrogenase belongs to subfamily 2 of the DMSO reductase-type molybdenum enzymes together with paralogous enzymes involved in the oxygen-independent hydroxylation of p-cymene, the isoprenoid side chains of sterols and even possibly n-alkanes; the subfamily also extends to dimethylsulfide dehydrogenases, selenite, chlorate and perchlorate reductases and, most significantly, dissimilatory nitrate reductases. The biochemical, spectroscopic and structural properties of the oxygen-independent hydroxylases among these enzymes are summarized and compared. All of them consist of three subunits, contain a molybdenum-bis-molybdopterin guanine dinucleotide cofactor, five Fe-S clusters and a heme b cofactor of unusual ligation, and are localized in the periplasmic space as soluble enzymes. In the case of ethylbenzene dehydrogenase, it has been determined that the heme b cofactor has a rather high redox potential, which may also be inferred for the paralogous hydroxylases. The known structure of ethylbenzene dehydrogenase allowed the calculation of detailed models of the reaction mechanism based on the density function theory as well as QM-MM (quantum mechanics - molecular mechanics) methods, which yield predictions of mechanistic properties such as kinetic isotope effects that appeared consistent with experimental data. PMID:26960184

  6. Structure-Based Inhibitor Design for Evaluation of a CYP3A4 Pharmacophore Model.

    Kaur, Parminder; Chamberlin, A Richard; Poulos, Thomas L; Sevrioukova, Irina F

    2016-05-12

    Human cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme that oxidizes and clears the majority of drugs. CYP3A4 inhibition may lead to drug-drug interactions, toxicity, and other adverse effects but, in some cases, could be beneficial and enhance therapeutic efficiency of coadministered pharmaceuticals that are metabolized by CYP3A4. On the basis of our investigations of analogs of ritonavir, a potent CYP3A4 inactivator and pharmacoenhancer, we have built a pharmacophore model for a CYP3A4-specific inhibitor. This study is the first attempt to test this model using a set of rationally designed compounds. The functional and structural data presented here agree well with the proposed pharmacophore. In particular, we confirmed the importance of a flexible backbone, the H-bond donor/acceptor moiety, and aromaticity of the side group analogous to Phe-2 of ritonavir and demonstrated the leading role of hydrophobic interactions at the sites adjacent to the heme and phenylalanine cluster in the ligand binding process. The X-ray structures of CYP3A4 bound to the rationally designed inhibitors provide deeper insights into the mechanism of the CYP3A4-ligand interaction. Most importantly, two of our compounds (15a and 15b) that are less complex than ritonavir have comparable submicromolar affinity and inhibitory potency for CYP3A4 and, thus, could serve as templates for synthesis of second generation inhibitors for further evaluation and optimization of the pharmacophore model. PMID:26371436

  7. The Nuclear Factor-kB Pathway Regulates Cytochrome P450 3A4 Protein Stability

    Zangar, Richard C.; Bollinger, Nikki; Verma, Seema; Karin, Norm J.; Lu, Yi

    2008-06-01

    We have previously observed that CYP3A4 protein levels are suppressed by inhibition of the proteasome in primary cultured hepatocytes. Because this result is opposite of what would be expected if CYP3A4 is degraded by the proteasome, it seems likely that there is another protein that is susceptible to proteasomal degradation that regulates CYP3A4 expression. In this study, we evaluate whether the nuclear factor kappa B (NF-kB) pathway is involved in that process. Our model system uses an adenovirus system to express CYP3A4 protein in HepG2 cells, which are derived from human cancer cells. Similar to results in primary hepatocytes, we found that inhibition of the proteasome with MG132 suppresses CYP3A4. Consistent with reports that proteasome inhibition suppresses the NF-kB pathway, we also observe a suppression of inhibitory kB kinase protein levels after treatment with MG132. Treatment of the HepG2 cells with NK-kB Activation Inhibitor also suppresses CYP3A4 proteins levels. In contrast, inhibition of either the proteasome or NF-kB pathways increases CYP3A4 mRNA levels. When the HepG2 cells are treated with cycloheximide, a general inhibitor of translation, the loss of CYP3A4 protein is accelerated by co-treatment with an NF-kB Activation Inhibitor. These results indicate that NF-kB activity regulates CYP3A4 protein stability and suggest that the NF-kB pathway is responsible for the decrease in CYP3A4 protein levels that results from the inhibition of proteasomal activity.

  8. In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology

    Fang, Huan; Dong, Huina; Cai, Tao; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

    2016-01-01

    In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model’s predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals. PMID:26974652

  9. CYP3A4 and CYP3A5 genotyping by Pyrosequencing

    McLeod Howard L

    2005-05-01

    Full Text Available Abstract Background Human cytochrome P450 3A enzymes, particularly CYP3A4 and CYP3A5, play an important role in drug metabolism. CYP3A expression exhibits substantial interindividual variation, much of which may result from genetic variation. This study describes Pyrosequencing assays for key SNPs in CYP3A4 (CYP3A4*1B, CYP3A4*2, and CYP3A4*3 and CYP3A5 (CYP3A5*3C and CYP3A5*6. Methods Genotyping of 95 healthy European and 95 healthy African volunteers was performed using Pyrosequencing. Linkage disequilibrium, haplotype inference, Hardy-Weinberg equilibrium, and tag SNPs were also determined for these samples. Results CYP3A4*1B allele frequencies were 4% in Europeans and 82% in Africans. The CYP3A4*2 allele was found in neither population sample. CYP3A4*3 had an allele frequency of 2% in Europeans and 0% in Africans. The frequency of CYP3A5*3C was 94% in Europeans and 12% in Africans. No CYP3A5*6 variants were found in the European samples, but this allele had a frequency of 16% in the African samples. Allele frequencies and haplotypes show interethnic variation, highlighting the need to analyze clinically relevant SNPs and haplotypes in a variety of ethnic groups. Conclusion Pyrosequencing is a versatile technique that could improve the efficiency of SNP analysis for pharmacogenomic research with the ultimate goal of pre-screening patients for individual therapy selection.

  10. Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus1

    De Luca, Vincenzo; Cutler, Adrian J.

    1987-01-01

    The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline. PMID:16665811

  11. The effect of complementary and alternative medicines on CYP3A4-mediated metabolism of three different substrates : 7-benzyloxy-4-trifluoromethyl-coumarin, midazolam and docetaxel

    Mooiman, Kim D; Maas-Bakker, Roel F; Hendrikx, Jeroen J M A; Bank, Paul C D; Rosing, Hilde; Beijnen, Jos H; Schellens, Jan H M; Meijerman, Irma

    2014-01-01

    OBJECTIVE: Concomitant use of complementary and alternative medicine (CAM) and anticancer drugs can affect the pharmacokinetics of anticancer drugs by inhibiting the metabolizing enzyme cytochrome P450 3A4 (CYP3A4) (EC 1.14.13.157). Several in vitro studies determined whether CAM can inhibit CYP3A4,

  12. Isolation and characterizacion of enzymes involved in the biosynthesis of secondary metabolites with phytotherapeutic interest

    Trapero Mozos, Almudena

    2013-01-01

    Isolation and characterization of enzymes involved in the biosynthesis of secondary metabolites with phytotherapeutic interest. Las plantas medicinales han sido utilizadas como primer recurso sanitario desde la antigüedad. En los últimos años el interés de la industria farmacéutica por los productos naturales o metabolitos secundarios provenientes de plantas medicinales, se ha incrementado considerablemente, entre las que se incluyen el azafrán. Se conoce como especia azafrán a los estigma...

  13. Endogenous enzymes involved in the transformation of oleuropein in Spanish table olive varieties.

    Ramírez, Eva; Medina, Eduardo; Brenes, Manuel; Romero, Concepción

    2014-10-01

    The main Spanish table olive varieties supplied by different olive cooperatives were investigated for their polyphenol compositions and the endogenous enzymes involved in their transformations during two growing seasons. Olives of the Manzanilla variety had the highest concentration in total polyphenols, followed by the Hojiblanca and Gordal varieties. The Gordal and Manzanilla cultivars showed the highest polyphenol oxidase activities. The Gordal cultivar presented a greater β-glucosidase and esterase activity than the others. An important influence of pH and temperature on the optimal activity of these enzymes was also observed. The polyphenol oxidase activity increased with temperature, and peroxidase activity was optimal at 35 °C. The β-glucosidase and esterase activities were at their maximum at 30 and 55 °C, respectively. The oxidase and β-glucosidase activities were at their maximum at the pH of the raw fruit. These results will contribute to the knowledge of the enzyme transformation of oleuropein in natural table olives. PMID:25209163

  14. Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans

    Shevelev Igor V

    2007-08-01

    Full Text Available Abstract Background Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. Results In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. Conclusion Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a

  15. A Cytochrome P450 3A4 Biosensor Based on Generation 4.0 PAMAM Dendrimers for the Detection of Caffeine.

    Müller, Michael; Agarwal, Neha; Kim, Jungtae

    2016-01-01

    Cytochromes P450 (CYP, P450) are a large family of heme-active-site proteins involved in many catalytic processes, including steroidogenesis. In humans, four primary enzymes are involved in the metabolism of almost all xenobiotics. Among these enzymes, CYP3A4 is responsible for the inactivation of the majority of used drugs which makes this enzyme an interesting target for many fields of research, especially pharmaceutical research. Since the late 1970s, attempts have been made to construct and develop electrochemical sensors for the determination of substrates. This paper is concerned with the establishment of such a CYP3A4-containing biosensor. The sensor was constructed by adsorption of alternating layers of sub-nanometer gold particle-modified PAMAM (poly-amido-amine) dendrimers of generation 4.0, along with the enzyme by a layer-by-layer assembly technique. Atomic force microscopy (AFM), quartz crystal microbalance (QCM), and Fourier-transformed infrared spectroscopy (FTIR) were employed to elucidate the sensor assembly. Additionally, the biosensor was tested by cyclic voltammetry using caffeine as a substrate. PMID:27548239

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

    Li-Na Gao

    2015-01-01

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

  17. Enzymes involved in vinyl acetate decomposition by Pseudomonas fluorescens PCM 2123 strain.

    Szczyrba, Elżbieta; Greń, Izabela; Bartelmus, Grażyna

    2014-03-01

    Esterases are widely used in food processing industry, but there is little information concerning enzymes involved in decompositions of esters contributing to pollution of environment. Vinyl acetate (an ester of vinyl alcohol and acetic acid) is a representative of volatile organic compounds (VOCs) in decomposition, of which hydrolyses and oxidoreductases are mainly involved. Their activities under periodically changing conditions of environment are essential for the removal of dangerous VOCs. Esterase and alcohol/aldehyde dehydrogenase activities were determined in crude cell extract from Pseudomonas fluorescens PMC 2123 after vinyl acetate induction. All examined enzymes exhibit their highest activity at 30-35 °C and pH 7.0-7.5. Esterase preferably hydrolyzed ester bonds with short fatty chains without plain differences for C2 or C4. Comparison of Km values for alcohol and aldehyde dehydrogenases for acetaldehyde suggested that this metabolite was preferentially oxidized than reduced. Activity of alcohol dehydrogenase reducing acetaldehyde to ethanol suggested that one mechanism of defense against the elevated concentration of toxic acetaldehyde could be its temporary reduction to ethanol. Esterase activity was inhibited by phenylmethanesulfonyl fluoride, while β-mercaptoethanol, dithiothreitol, and ethylenediaminetetraacetic acid had no inhibitor effect. From among metal ions, only Mg(2+) and Fe(2+) stimulated the cleavage of ester bond. PMID:23913099

  18. Exploration of soil metagenome diversity for prospection of enzymes involved in lignocellulosic biomass conversion

    Alvarez, T.M.; Squina, F.M. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Paixao, D.A.A.; Franco Cairo, J.P.L.; Buchli, F.; Ruller, R. [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Campinas, SP (Brazil); Prade, R. [Oklahoma State University, Sillwater, OK (United States)

    2012-07-01

    Full text: Metagenomics allows access to genetic information encoded in DNA of microorganisms recalcitrant to cultivation. They represent a reservoir of novel biocatalyst with potential application in environmental friendly techniques aiming to overcome the dependence on fossil fuels and also to diminish air and water pollution. The focus of our work is the generation of a tool kit of lignocellulolytic enzymes from soil metagenome, which could be used for second generation ethanol production. Environmental samples were collected at a sugarcane field after harvesting, where it is expected that the microbial population involved on lignocellulose degradation was enriched due to the presence of straws covering the soil. Sugarcane Bagasse-Degrading-Soil (SBDS) metagenome was massively-parallel-454-Roche-sequenced. We identified a full repertoire of genes with significant match to glycosyl hydrolases catalytic domain and carbohydrate-binding modules. Soil metagenomics libraries cloned into pUC19 were screened through functional assays. CMC-agar screening resulted in positive clones, revealing new cellulases coding genes. Through a CMC-zymogram it was possible to observe that one of these genes, nominated as E-1, corresponds to an enzyme that is secreted to the extracellular medium, suggesting that the cloned gene carried the original signal peptide. Enzymatic assays and analysis through capillary electrophoresis showed that E-1 was able to cleave internal glycosidic bonds of cellulose. New rounds of functional screenings through chromogenic substrates are being conducted aiming the generation of a library of lignocellulolytic enzymes derived from soil metagenome, which may become key component for development of second generation biofuels. (author)

  19. Exploration of soil metagenome diversity for prospection of enzymes involved in lignocellulosic biomass conversion

    Full text: Metagenomics allows access to genetic information encoded in DNA of microorganisms recalcitrant to cultivation. They represent a reservoir of novel biocatalyst with potential application in environmental friendly techniques aiming to overcome the dependence on fossil fuels and also to diminish air and water pollution. The focus of our work is the generation of a tool kit of lignocellulolytic enzymes from soil metagenome, which could be used for second generation ethanol production. Environmental samples were collected at a sugarcane field after harvesting, where it is expected that the microbial population involved on lignocellulose degradation was enriched due to the presence of straws covering the soil. Sugarcane Bagasse-Degrading-Soil (SBDS) metagenome was massively-parallel-454-Roche-sequenced. We identified a full repertoire of genes with significant match to glycosyl hydrolases catalytic domain and carbohydrate-binding modules. Soil metagenomics libraries cloned into pUC19 were screened through functional assays. CMC-agar screening resulted in positive clones, revealing new cellulases coding genes. Through a CMC-zymogram it was possible to observe that one of these genes, nominated as E-1, corresponds to an enzyme that is secreted to the extracellular medium, suggesting that the cloned gene carried the original signal peptide. Enzymatic assays and analysis through capillary electrophoresis showed that E-1 was able to cleave internal glycosidic bonds of cellulose. New rounds of functional screenings through chromogenic substrates are being conducted aiming the generation of a library of lignocellulolytic enzymes derived from soil metagenome, which may become key component for development of second generation biofuels. (author)

  20. Study Liver Cytochrome P450 3A4 Inhibition and Hepatotoxicity Using DMSO-Differentiated HuH-7 Cells.

    Liu, Yitong

    2016-01-01

    Metabolically competent, inexpensive, and robust in vitro cell models are needed for studying liver drug-metabolizing enzymes and hepatotoxicity. Human hepatoma HuH-7 cells develop into a differentiated in vitro model resembling primary human hepatocytes after a 2-week dimethyl sulfoxide (DMSO) treatment. DMSO-treated HuH-7 cells express elevated cytochrome P450 3A4 (CYP3A4) enzyme gene expression and activity compared to untreated HuH-7 cells. This cell model could be used to study CYP3A4 inhibition by reversible and time-dependent inhibitors, including drugs, food-related substances, and environmental chemicals. The DMSO-treated HuH-7 model is also a suitable tool for investigating hepatotoxicity. This chapter describes a detailed methodology for developing DMSO-treated HuH-7 cells, which are subsequently used for CYP3A4 inhibition and hepatotoxicity studies. PMID:27518624

  1. A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4

    Zi-Ru Dai

    2015-06-01

    Full Text Available Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s and the site(s of modification. The newly established model was applied to predict the metabolic site(s of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s of CYP3A4 on steroids with high predictive accuracy.

  2. Trichostatin A, a histone deacetylase inhibitor stimulate CYP3A4 proximal promoter activity in Hepa-I cells.

    Ahn, Mee Ryung; Kim, Dae-Kee; Sheen, Yhun Yhong

    2004-04-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant CYPs in human liver, comprising approximately 30% of the total liver CYPs contents and is involved in the metabolism of more than 60% of currently used therapeutic drugs. However, the molecular mechanisms underlying regulation of CYP3A4 gene expression have not been understood. Thus, this study has been carried out to gain the insight of the molecular mechanism of CYP3A4 gene expression, investigating if the histone deacetylation is involved in the regulation of CYP3A4 gene expression by proximal promoter. Also SXR was investigated to see if they were involved in the regulation of CYP3A4 proximal promoter activity. Hepa-I cells were transfected with a plasmid containing approximately 1 kb of the human CYP3A4 proximal promoter region (863 to +64 bp) cloned in front of a reporter gene, luciferase, in the presence or absence of SXR. Transfected cells were treated with CYP3A4 inducers such as rifampicin, PCN and RU 486, in order to examine the regulation of CYP3A4 gene expression in the presence or absence of trichostatin A (TSA). In Hepa-I cells, CYP3A4 inducers increased modestly the luciferase activity when TSA was co-treated, but this increment was not enhanced by SXR cotransfection. Taken together, these results indicated that the inhibition of histone deacetylation was required to SXR-mediated increase in CYP3A4 proximal promoter region when rifampicin, or PCN was treated. Further a trans-activation by SXR may demand other species-specific transcription factors. PMID:15180307

  3. Identification of a functional homolog of the mammalian CYP3A4 in locusts.

    Olsen, Line Rørbæk; Gabel-Jensen, Charlotte; Nielsen, Peter Aadal; Hansen, Steen Honoré; Badolo, Lassina

    2014-07-01

    Insects have been proposed as a new tool in early drug development. It was recently demonstrated that locusts have an efflux transporter localized in the blood-brain barrier (BBB) that is functionally similar to the mammalian P-glycoprotein efflux transporter. Two insect BBB models have been put forward, an ex vivo model and an in vivo model. To use the in vivo model it is necessary to fully characterize the locust as an entire organism with regards to metabolic pathways and excretion rate. In the present study, we have characterized the locust metabolism of terfenadine, a compound that in humans is specific to the cytochrome P450 enzyme 3A4. Using high-resolution mass spectrometry coupled to ultra-high-performance liquid chromatography, we have detected metabolites identical to human metabolites of terfenadine. The formation of human metabolites in locusts was inhibited by ketoconazole, a mammalian CYP3A4 inhibitor, suggesting that the enzyme responsible for the human metabolite formation in locusts is functionally similar to human CYP3A4. Besides the human metabolites of terfenadine, additional metabolites were formed in locusts. These were tentatively identified as phosphate and glucose conjugates. In conclusion, not only may locusts be a model useful for determining BBB permeation, but possibly insects could be used in metabolism investigation. However, extensive characterization of the insect model is necessary to determine its applicability. PMID:24778367

  4. Imatinib treatment and pharmacogenotype CYP3A4 in relation with the clonal expansion Ph(+ in chronic myeloid leukemia (CML.

    Mauricio Camargo

    2009-12-01

    Full Text Available Introduction: Imatinib is an inhibitor of the BCR-ABL tyrosine-kinase that has dramatically changed the treatment of patient with Chronic myeloid leukemia (CML positive for the Philadelphia chromosome (Ph+. This compound is mainly metabolized by the cytochrome CYP3A4 enzyme, coded by a gene with individual variations that could interfere with the effectiveness of the treatment, due to the fact that particular single nucleotide polymorphisms (SNPs, i.e., CYP3A4*1B y CYP3A4*2, have shown to exert a significant influence on the metabolic activity of this pharmacologically important enzyme. Objective: Evaluate the frequency of pharmacogenetically important polymorphisms in the CYP3A4 gen in a Colombian population of patients with CML being treated with this novel drug (Imatinib, in parallel with a control population of 164 healthy individuals. Correlate the evolution of the clonal expansion Ph(+ with the presence of these SNPs and the length of treatment. Methodology: PCR-RFLP genotyping for the CYP3A4* 1B y CYP3A4*2 SNPs. RBHG replication banding for the evaluation of the presence of the Ph(+ markers in spontaneous mitotic blasts. Results: A positive cytogenetic response and/or correlation was detected between the length of the imatinib treatment and a reduction in the percentage of Ph(+ blasts. Genotyping indicate that CYP3A4*1B polymorphism does no affect the cytogenetic response in imatinib treated Ph(+ patients, and that the pharmacorelevant CYP3A4*2 SNP is not present in this population of patients and controls (N=194. Conclusions: The pharmacogenotype CYP3A4*2 (exon 7 does not affect the induced positive cytogenetic response triggered by the imatinib treatment, that generally induces a reduction in Ph(+ blasts en relation with the duration of the treatment.

  5. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets

    Pinto-Fernandez, Adan; Kessler, Benedikt M.

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  6. Flavonoids activate pregnane × receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells

    Wu Jing

    2010-06-01

    Full Text Available Abstract Background The expression of the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4 is regulated by the pregnane × receptor (PXR, which is modulated by numerous signaling pathways, including the cyclin-dependent kinase (Cdk pathway. Flavonoids, commonly consumed by humans as dietary constituents, have been shown to modulate various signaling pathways (e.g., inhibiting Cdks. Flavonoids have also been shown to induce CYPs expression, but the underlying mechanism of action is unknown. Here, we report the mechanism responsible for flavonoid-mediated PXR activation and CYP expression. Results In a cell-based screen designed to identify compounds that activate PXR-mediated CYP3A4 gene expression in HepG2 human carcinoma cells, we identified several flavonoids, such as luteolin and apigenin, as PXR activators. The flavonoids did not directly bind to PXR, suggesting that an alternative mechanism may be responsible for flavonoid-mediated PXR activation. Consistent with the Cdk5-inhibitory effect of flavonoids, Cdk5 and p35 (a non-cyclin regulatory subunit required to activate Cdk5 were expressed in HepG2. The activation of Cdk5 attenuated PXR-mediated CYP3A4 expression whereas its downregulation enhanced it. The Cdk5-mediated downregulation of CYP3A4 promoter activity was restored by flavonoids, suggesting that flavonoids activate PXR by inactivating Cdk5. In vitro kinase assays showed that Cdk5 directly phosphorylates PXR. The Cdk kinase profiling assay showed that apigenin inhibits multiple Cdks, suggesting that several Cdks may be involved in activation of PXR by flavonoids. Conclusions Our results for the first time link the stimulatory effect of flavonoids on CYP expression to their inhibitory effect on Cdks, through a PXR-mediated mechanism. These results may have important implications on the pharmacokinetics of drugs co-administered with herbal remedy and herbal-drug interactions.

  7. Hepatic fatty acid oxidation : activity, localization and function of some enzymes involved

    A. van Tol (Arie)

    1971-01-01

    textabstractFatty acid oxidation is an important pathway for energy production in mammals and birds. In animal tissues the enzymes of fatty acid oxidation are located in the mitochondrion. Recent reports suggest that this is not the case in Castor bean endosperm. In this tissue the enzymes of B-oxid

  8. A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

    Moriya, Shunsuke; Iwasaki, Kaori [Department of Molecular Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Samejima, Keijiro, E-mail: samejima-kj@igakuken.or.jp [Department of Molecular Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Takao, Koichi [Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295 (Japan); Kohda, Kohfuku [Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo, Tokyo 202-8585 (Japan); Hiramatsu, Kyoko; Kawakita, Masao [Department of Molecular Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506 (Japan)

    2012-10-20

    Highlights: Black-Right-Pointing-Pointer Compounds in polyamine catabolic pathway were determined by a column-free ESI-TOF MS. Black-Right-Pointing-Pointer N{sup 1}- and N{sup 8}-acetylspermidine were determined by a column-free ESI-MS/MS. Black-Right-Pointing-Pointer The method was applied to determine activities of APAO, SMO, and SSAT in the pathway. Black-Right-Pointing-Pointer The assay method contained stable isotope-labeled natural substrates. Black-Right-Pointing-Pointer It is applicable to biological samples containing natural substrate and product. - Abstract: An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N{sup 1}-acetylspermidine (N{sup 1}AcSpd), N{sup 8}-acetylspermidine (N{sup 8}AcSpd), N{sup 1}-acetylspermine, N{sup 1},N{sup 8}-diacetylspermidine, and N{sup 1},N{sup 12}-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N{sup 1}AcSpd and N{sup 8}AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with {sup 13}C{sub 2}-N{sup 1}AcSpd and {sup 13}C{sub 2}-N{sup 8}AcSpd which have the {sup 13}C{sub 2}-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N{sup 1}-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N{sup 1}-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-{sup 15}N{sub 3}]-N{sup 1}-acetylspermine and [1,4,8-{sup 15}N{sub 3

  9. Mixing apples and oranges: Analysis of heterotropic cooperativity in cytochrome P450 3A4.

    Frank, Daniel J; Denisov, Ilia G; Sligar, Stephen G

    2009-08-15

    Heterotropic cooperative phenomena have been documented in studies with cytochrome P450 3A4, with few attempts to quantify this behavior other than to show the apparent stimulatory effect of certain CYP3A4 substrates on the enzyme's catalytic activity for others. Here CYP3A4 solubilized in Nanodiscs is studied for its ability to interact with two substrates, alpha-naphthoflavone and testosterone, which produce transitions in the heme spin state with apparent spectral affinities (corrected for membrane partitioning) of 7 and 38 microM, respectively. Simultaneous addition of both substrates at fixed molar ratios allows for the separation of specific heterotropic cooperative interactions from the simple additive affinities for the given substrate ratios. The absence of any changes in the normalized spectral dissociation constant due to changes in substrate ratio reveals that the observed stimulatory effect is largely due to differences in the relative substrate affinities and the presence of additional substrate in the system, rather than any specific positive heterotropic interactions between the two substrates. PMID:19560436

  10. Enzymes involved in 3,5-diaminohexanoate degradation by Brevibacterium sp.

    Barker, H. A.; Kahn, J. M.; Chew, S

    1980-01-01

    Cell-free extracts of Brevibacterium sp. L5 grown on DL-erythro-3,5-diaminohexanoate were found to contain a 3-keto-5-aminohexanoate cleavage enzyme that converts 3-keto-5-aminohexanoate and acetyl-coenzyme A (CokA) to 3-aminobutyryl-CoA and acetoacetate and a deaminase that coverts L-3-aminobutyryl-CoA to crotonyl-CoA. The cleavage enzyme has been purified extensively, and some of its properties have been determined for comparison with the 3-keto-6-acetamido-hexanoate cleavage enzyme of Pseu...

  11. Developmental changes in enzymes involved in the conversion of hexose phosphate and its subsequent metabolites during early tuberization of potato

    Appeldoorn, N.J.G.; Bruijn, de S.M.; Koot-Gronsveld, E.A.M.; Visser, R.G.F.; Vreugdenhil, D.; Plas, van der L.H.W.

    1999-01-01

    A highly synchronized in vitro tuberization system, based on single-node cuttings containing an axillary bud, was used to investigate the activity patterns of enzymes involved in the conversion of hexose phosphates and related products during stolon-to-tuber transition of potato (Solanum tuberosum L

  12. Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir.

    Kumar, Santosh; Earla, Ravinder; Jin, Mengyao; Mitra, Ashim K; Kumar, Anil

    2010-11-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K(D) (5.9±0.34mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20mM) decreased the K(D) of nelfinavir by >5-fold (0.041±0.007 vs. 0.227±0.038μM). Similarly, 20mM ethanol decreased the IC(50) of nelfinavir by >3-fold (2.6±0.5 vs. 8.3±3.1μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k(cat), it decreased the K(m) of nelfinavir, suggesting a decrease in catalytic efficiency (k(cat)/K(m)). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy. PMID:20937259

  13. Regulation of genes encoding enzymes involved in plant cell wall deconstruction in Trichoderma reesei

    Ries, Laure Nicolas Annick

    2013-01-01

    This study describes the regulation of genes encoding plant cell wall-degrading enzymes in the presence of different carbon sources from the biotechnologically important fungus Trichoderma reesei. It was shown that different carbon sources influence fungal growth rate, biomass production and subsequent enzyme secretion. Several genes were identified and suggested to play a role in the development of conidia and in maintaining polarised growth. RNA-sequencing studies showed an increase in t...

  14. High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

    Gregory Kohn

    Full Text Available BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i, which could be cytotoxic because of its high affinity for Ca(2+, thereby interfering with Ca(2+ signaling.

  15. Genetic variability in CYP3A4 and CYP3A5 in primary liver, gastric and colorectal cancer patients

    García Monserrat

    2007-07-01

    Full Text Available Abstract Background Drug-metabolizing enzymes play a role in chemical carcinogenesis through enzymatic activation of procarcinogens to biologically reactive metabolites. The role of gene polymorphisms of several cytochrome P450 enzymes in digestive cancer risk has been extensively investigated. However, the drug-metabolizing enzymes with the broader substrate specificity, CYP3A4 and CYP3A5, have not been analyzed so far. This study aims to examine associations between common CYP3A4 and CYP3A5 polymorphisms and digestive cancer risk. Methods CYP3A4 and CYP3A5 genotypes were determined in 574 individuals including 178 patients with primary liver cancer, 82 patients with gastric cancer, 151 patients with colorectal cancer, and 163 healthy individuals. Results The variant allele frequencies for patients with liver cancer, gastric cancer, colorectal cancer and healthy controls, respectively, were: CYP3A4*1B, 4.8 % (95% C.I. 2.6–7.0, 3.7 % (0.8–6.6 4.3% (2.0–6.6 and 4.3% (2.1–6.5; CYP3A5*3, 91.8 % (93.0–97.4, 95.7% (92.6–98.8, 91.7% (88.6–94.8 and 90.8% (87.7–93.9. The association between CYP3A4*1B and CYP3A5*3 variant alleles did not significantly differ among patients and controls. No differences in genotypes, allele frequencies, or association between variant alleles were observed with regard to gender, age at diagnosis, tumour site or stage. Conclusion Common polymorphisms on CYP3A4 and CYP3A5 genes do not modify the risk of developing digestive cancers in Western Europe.

  16. In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4

    Qiao-Li Lv

    2015-12-01

    Full Text Available Glycyrrhetinic acid (GA has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450 cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC50 values of 8.195 μM in HLMs and 7.498 μM in the recombinant cDNA-expressed CYP3A4 enzyme system, respectively. It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver–Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 μM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 μM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4.

  17. Involvement of detergent-insoluble complexes in the intracellular transport of intestinal brush border enzymes

    Danielsen, E M

    1995-01-01

    A number of transmembrane digestive enzymes of the porcine small intestinal brush border membrane were found to be partially Triton X-100-insoluble at 0 degree C and colocalized in gradient centrifugation experiments with the GPI-anchored alkaline phosphatase in low-density, detergent......%), and lactase-phlorizin hydrolase (EC 3.2.1.23-62) was essentially fully soluble in detergent. In radioactively labeled, mucosal explants, the newly synthesized brush border enzymes began to associate with detergent-insoluble complexes while still in their transient, high mannose-glycosylated form, and...... their insolubility increased to that of the steady-state level soon after they achieved their mature, complex glycosylation, i.e., after passage through the Golgi complex. Detergent-insoluble complexes isolated by density gradient centrifugation were highly enriched in brush border enzymes, and the...

  18. Phenotype-genotype variability in the human CYP3A locus as assessed by the probe drug quinine and analyses of variant CYP3A4 alleles

    The human cytochrome P450 3A (CYP3A) enzymes, which metabolize 50% of currently used therapeutic drugs, exhibit great interindividual differences in activity that have a major impact on drug treatment outcome, but hitherto no genetic background importantly contributing to this variation has been identified. In this study we show that CYP3A4 mRNA and hnRNA contents with a few exceptions vary in parallel in human liver, suggesting that mechanisms affecting CYP3A4 transcription, such as promoter polymorphisms, are relevant for interindividual differences in CYP3A4 expression. Tanzanian (n = 143) healthy volunteers were phenotyped using quinine as a CYP3A probe and the results were used for association studies with CYP3A4 genotypes. Carriers of CYP3A4*1B had a significantly lower activity than those with CYP3A4*1 whereas no differences were seen for five other SNPs investigated. Nuclear proteins from the B16A2 hepatoma cells were found to bind with less affinity to the CYP3A4*1B element around -392 bp as compared to CYP3A4*1. The data indicate the existence of a genetic CYP3A4 polymorphism with functional importance for interindividual differences in enzyme expression

  19. Phycobiliprotein biosynthesis in cyanobacteria: structure and function of enzymes involved in post-translational modification.

    Schluchter, Wendy M; Shen, Gaozhong; Alvey, Richard M; Biswas, Avijit; Saunée, Nicolle A; Williams, Shervonda R; Mille, Crystal A; Bryant, Donald A

    2010-01-01

    Cyanobacterial phycobiliproteins are brilliantly colored due to the presence of covalently attached chromophores called bilins, linear tetrapyrroles derived from heme. For most phycobiliproteins, these post-translational modifications are catalyzed by enzymes called bilin lyases; these enzymes ensure that the appropriate bilins are attached to the correct cysteine residues with the proper stereochemistry on each phycobiliprotein subunit. Phycobiliproteins also contain a unique, post-translational modification, the methylation of a conserved asparagine (Asn) present at beta-72, which occurs on the beta-subunits of all phycobiliproteins. We have identified and characterized several new families of bilin lyases, which are responsible for attaching PCB to phycobiliproteins as well as the Asn methyl transferase for beta-subunits in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. All of the enzymes responsible for synthesis of holo-phycobiliproteins are now known for this cyanobacterium, and a brief discussion of each enzyme family and its role in the biosynthesis of phycobiliproteins is presented here. In addition, the first structure of a bilin lyase has recently been solved (PDB ID: 3BDR). This structure shows that the bilin lyases are most similar to the lipocalin protein structural family, which also includes the bilin-binding protein found in some butterflies. PMID:20532743

  20. Hydroxylation of 20-hydroxyvitamin D3 by human CYP3A4.

    Cheng, Chloe Y S; Slominski, Andrzej T; Tuckey, Robert C

    2016-05-01

    20S-Hydroxyvitamin D3 [20(OH)D3] is the biologically active major product of the action of CYP11A1 on vitamin D3 and is present in human plasma. 20(OH)D3 displays similar therapeutic properties to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], but without causing hypercalcaemia and therefore has potential for development as a therapeutic drug. CYP24A1, the kidney mitochondrial P450 involved in inactivation of 1,25(OH)2D3, can hydroxylate 20(OH)D3 at C24 and C25, with the products displaying more potent inhibition of melanoma cell proliferation than 20(OH)D3. CYP3A4 is the major drug-metabolising P450 in liver endoplasmic reticulum and can metabolise other active forms of vitamin D, so we examined its ability to metabolise 20(OH)D3. We found that CYP3A4 metabolises 20(OH)D3 to three major products, 20,24R-dihydroxyvitamin D3 [20,24R(OH)2D3], 20,24S-dihydroxyvitamin D3 [20,24S(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. 20,24R(OH)2D3 and 20,24S(OH)2D3, but not 20,25(OH)2D3, were further metabolised to trihydroxyvitamin D3 products by CYP3A4 but with low catalytic efficiency. The same three primary products, 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3, were observed for the metabolism of 20(OH)D3 by human liver microsomes, in which CYP3A4 is a major CYP isoform present. Addition of CYP3A family-specific inhibitors, troleandomycin and azamulin, almost completely inhibited production of 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3 by human liver microsomes, further supporting that CYP3A4 plays the major role in 20(OH)D3 metabolism by microsomes. Since both 20,24R(OH)2D3 and 20,25(OH)2D3 have previously been shown to display enhanced biological activity in inhibiting melanoma cell proliferation, our results show that CYP3A4 further activates, rather than inactivates, 20(OH)D3. PMID:26970587

  1. Extraction of pectic enzymes from of Lulo (Solanum quitoense lam) involved in softening

    The main problem of post-harvest deterioration of Lulo (Solanum quitoense lam) is the softening is the main problem of post-harvest deterioration of Lulo that is generated mainly by the activity of pectic enzymes, which attack the structural network of the cell wall. this research was based on finding the best conditions structural cell wall network for extraction and measurement of enzyme activity pectinesterase (PE), polygalacturonase (PG) and pectato liasa (PL); tools needed to study the further role of these enzymes in the deterioration of pectatelyase fruit softening, due to various metabolic changes. It was found that the first two enzymes can be extracted simultaneously with 20 mm phosphate buffer pH 7.0, 0.06 m NaCl and 60 minutes of extraction, ratio 1:2 (plant material: extraction buffer), pectatelyase extracted with 20 mm phosphate buffer pH 7.0, 20 mm cysteine and 30 minutes of extraction, ratio 1:3. for quantification of pectinesterase activity is necessary to incubate 15 minutes at 42 Celsius degrade, 2500 μl of crude enzyme extract (EE) in 20 mm phosphate buffer pH 7.0, to 0.15 m NaCl and 1.6% citrus pectin as (CP) substrate with apparent km values of 3.78% CP and vmax 17.95 mol h+/min, mg prot. for the quantification of pectinesterase activity is necessary to incubate 15 minutes to 42 Celsius degrade 2500 μl of crude enzyme extract (EE) in 20 mm phosphate buffer pH 7.0, 0.15 m NaCl and 1.6% citrus pectin as substrate with apparent km values of 3.78% CP and 17.95 μ vmax mol h+/min Mg prot. for the quantification of polygalacturonase activity is necessary to incubate 15 minutes to 37 Celsius degrade 30 μl (EE) in 200 mm acetate buffer pH 4.5, 0.25 m NaCl and 1.0% of APG as substrate, with apparent km values 0.141% of APG and vmax 28.46 nkat/s mg prot. for the quantification of the pectatelyase activity is necessary to incubate 2 minutes to 17 Celsius degrade, 100 μl (EE) in buffer tris: HCl pH 8.5, 50 mm 4 mm CaCl2 and 0.1% PGA as substrate, with

  2. Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829

    Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy

    2016-01-01

    The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to i...

  3. Expression and purification of lipoprotein-associated phospholipase A2, a key enzyme involved in atherosclerosis

    Fu-jun ZHANG; Mao-jun CAI; Jing-kang SHEN; Yi-ping WANG

    2006-01-01

    Aim: To express and purify lipoprotein-associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors using the expressed Lp-PLA2. Methods: We cloned the full-length cDNA of Lp-PLA2 from differentiated THP-1 cells, and subcloned the cDNA into the baculovirus transfer vector pFastBacl. In addition, we introduced an N-terminal Kozak sequence for highlevel translation initiation and a C-terminal sequence of 6 histidine residues for purification. The fusion enzyme was expressed in Sf9 insect cells and purified by Ni2+ affinity chromatography. Recombinant Lp-PLA2 activity was measured using [3H]PAF as a substrate, and we examined the enzyme activity of recombinant Lp-PLA2 pretreated with the known Lp-PLA2 inhibitor SB435495. Results: We successfully cloned the full-length Lp-PLA2 gene from differentiated THP-1 cells. The fusion enzyme was expressed in Sf9 insect cells at a high level and purified efficiently through a 2-step procedure. The recombinant Lp-PLA2 activity was measured using [3H]PAF as a substrate, and proved to be enzymatically active. Lp-PLA2 inhibitor SB435495 produced a good inhibition curve for inhibition of recombinant Lp-PLA2 with an IC50 of 57±1 μmol/L. Conclusion: We expressed and purified Lp-PLA2 at a high level in insect cell-baculovirus expression system. The yield ratio was much greater than that obtained from human plasma and we established a screening model for Lp-PLA2 inhibitors using the expressed Lp-PLA2.

  4. Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

    Horchani, Faouzi; Aschi-Smiti, Samira

    2010-01-01

    In order to investigate the effects of root hypoxia (1–2% oxygen) on the nitrogen (N) metabolism of tomato plants (Solanum lycopersicum L. cv. Micro-Tom), a range of N compounds and N-assimilating enzymes were performed on roots and leaves of plants submitted to root hypoxia at the second leaf stage for three weeks. Obtained results showed that root hypoxia led to a significant decrease in dry weight (DW) production and nitrate content in roots and leaves. Conversely, shoot to root DW ratio a...

  5. Involvement of anti-oxidative enzymes, photosynthetic pigments and flavonoid metabolism in the adaptation of Reaumuria soongorica to salt stress

    YuBing Liu; Bo Cao; MeiLing Liu

    2013-01-01

    Reaumuria soongorica is a short woody shrub widely found in semi-arid areas of China. It can survive severe environ-mental stress including high salinity in its natural habitat. Thus, we investigated the involvement of anti-oxidative enzymes, photosynthetic pigments and flavonoid metabolism in the adaptation of R. soongorica to saline environments. R. soon-gorica was treated with 0, 100, 200 and 400 mM NaCl solutions for 14 days. Soil salt content increased significantly by watering with high content of NaCl solution, and no variation between 8 and 14 days during treatment. The levels of pe-roxidation of lipid membranes (measured by malondialdehyde content) and the activities of three antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX)) increased under salt stress. Chlorophyll and carotenoid content decreased with increasing salt content. The ratio of Chl a/Chl b and carotenoid/Chl exhibited sig-nificant increase under 400 mM NaCl. However, total flavonoid and anthocyanin contents and key enzyme activities in the flavonoid pathway including phenylalanine ammonialyase (PAL) and Chalcone isomerase (CHI) decreased under salt stress. These findings possibly suggest that R. soongorica has an adaptation protection mechanism against salt-induced oxidative damage by inducing the activity of antioxidant enzymes and maintaining a steady level of carotenoid/Chl.

  6. Investigating the binding interactions of the anti-Alzheimer's drug donepezil with CYP3A4 and P-glycoprotein.

    McEneny-King, Alanna; Edginton, Andrea N; Rao, Praveen P N

    2015-01-15

    The anti-Alzheimer's agent donepezil is known to bind to the hepatic enzyme CYP3A4, but its relationship with the efflux transporter P-glycoprotein (P-gp) is not as well elucidated. We conducted in vitro inhibition studies of donepezil using human recombinant CYP3A4 and P-gp. These studies show that donepezil is a weak inhibitor of CYP3A4 (IC50=54.68±1.00μM) whereas the reference agent ketoconazole exhibited potent inhibition (CYP3A4 IC50=0.20±0.01μM). P-gp inhibition studies indicate that donepezil exhibits better inhibition relative to CYP3A4 (P-gp EC50=34.85±4.63μM) although it was less potent compared to ketoconazole (P-gp EC50=9.74±1.23μM). At higher concentrations, donepezil exhibited significant inhibition of CYP3A4 (69%, 84% and 87% inhibition at 100, 250 and 500μM, respectively). This indicates its potential to cause drug-drug interactions with other CYP3A4 substrates upon co-administration; however, this scenario is unlikely in vivo due to the low therapeutic concentrations of donepezil. Similarly, donepezil co-administration with P-gp substrates or inhibitors is unlikely to result in beneficial or adverse drug interactions. The molecular docking studies show that the 5,6-dimethoxyindan-1-one moiety of donepezil was oriented closer to the heme center in CYP3A4 whereas in the P-gp binding site, the protonated benzylpiperidine pharmacophore of donepezil played a major role in its binding ability. Energy parameters indicate that donepezil complex with both CYP3A4 and P-gp was less stable (CDOCKER energies=-15.05 and -4.91kcal/mol, respectively) compared to the ketoconazole-CYP3A4 and P-gp complex (CDOCKER energies=-41.89 and -20.03kcal/mol, respectively). PMID:25499431

  7. Enzyme-linked immunosorbant assays for glucosidases involved in processing of N-linked glycoproteins

    The conventional biological assays for glucosidase I and II are to determine the release of 14C-glucose from radiolabeled Glc3Man9(GlcNAc)2 and Glc/sub 1-2/Man9(GlcNAc)2, respectively by the actions of glucosidases. They have developed ultrasensitive ELISA's for these two glycosidases by using highly specific polyclonal antibodies prepared against purified glucosidase I and II from the bovine mammary tissue. Anti-glucosidase I and anti-glucosidase II antibodies are shown to be monospecific and are cross-reactive towards glucosidase I and II from liver, mammary gland, kidney and heart tissues of cow, guinea pig, mouse and rat, respectively. Western blot analysis revealed that anti-glucosidase I and anti-glucosidase II are very specific and are not cross-reactive towards any other protein present in solubilized microsomal extracts. Using these antibodies, conditions for ELISA's for glucosidase I and II were standardized. Dose response curves for purified enzymes and crude extracts were developed. A correlation between biological activities of these enzymes and immunoreactive glucosidase I and II, present in the solubilized microsomal preparations of liver, mammary gland, kidney and heart tissues from cow, guinea pig, mouse and rat, is made. The sensitivity of ELISA's for glucosidase I and II are in the range of 10 - 1000 ng of immunoreactive species

  8. Evolutionary History of the Enzymes Involved in the Calvin-Benson Cycle in Euglenids.

    Markunas, Chelsea M; Triemer, Richard E

    2016-05-01

    Euglenids are an ancient lineage that may have existed as early as 2 billion years ago. A mere 65 years ago, Melvin Calvin and Andrew A. Benson performed experiments on Euglena gracilis and elucidated the series of reactions by which carbon was fixed and reduced during photosynthesis. However, the evolutionary history of this pathway (Calvin-Benson cycle) in euglenids was more complex than Calvin and Benson could have imagined. The chloroplast present today in euglenophytes arose from a secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga. A long period of evolutionary time existed before this secondary endosymbiotic event took place, which allowed for other endosymbiotic events or gene transfers to occur prior to the establishment of the green chloroplast. This research revealed the evolutionary history of the major enzymes of the Calvin-Benson cycle throughout the euglenid lineage and showed that the majority of genes for Calvin-Benson cycle enzymes shared an ancestry with red algae and/or chromophytes suggesting they may have been transferred to the nucleus prior to the acquisition of the green chloroplast. PMID:26566594

  9. Eucalyptus ESTs involved in the production of 9-cis epoxycarotenoid dioxygenase, a regulatory enzyme of abscisic acid production

    Iraê A. Guerrini

    2005-01-01

    Full Text Available Abscisic acid (ABA regulates stress responses in plants, and genomic tools can help us to understand the mechanisms involved in that process. FAPESP, a Brazilian research foundation, in association with four private forestry companies, has established the FORESTs database (https://forests.esalq.usp.br. A search was carried out in the Eucalyptus expressed sequence tag database to find ESTs involved with 9-cis epoxycarotenoid dioxygenase (NCED, the regulatory enzyme for ABA biosynthesis, using the basic local BLAST alignment tool. We found four clusters (EGEZLV2206B11.g, EGJMWD2252H08.g, EGBFRT3107F10.g, and EGEQFB1200H10.g, which represent similar sequences of the gene that produces NCED. Data showed that the EGBFRT3107F10.g cluster was similar to the maize (Zea mays NCED enzyme, while EGEZLV2206B11.g and EGJMWD2252H08.g clusters were similar to the avocado (Persea americana NCED enzyme. All Eucalyptus clusters were expressed in several tissues, especially in flower buds, where ABA has a special participation during the floral development process.

  10. Metabolic Activation of Pradefovir by CYP3A4 and Its Potential as an Inhibitor or Inducer

    Lin, Chin-Chung; Fang, Che; Benetton, Salete; Xu, Gui-fen; Yeh, Li-Tain

    2006-01-01

    Metabolic activation of pradefovir to 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was evaluated by using cDNA-expressed CYP isozymes in portal vein-cannulated rats following oral administration and in human liver microsomes. The enzyme induction potential of pradefovir was evaluated in rats following multiple oral dosing and in primary cultures of human hepatocytes. The results indicated that CYP3A4 is the only cDNA-expressed CYP isozyme catalyzing the conversion of pradefovir to PMEA. Pradefo...

  11. Erectogenic and Aphrodisiac Effects of Butea frondosa Koenig ex Roxb. in Rats: Involvement of Enzyme Inhibition

    Sumanta Kumar Goswami

    2013-01-01

    Full Text Available Butea frondosa Koenig ex Roxb. (BF is traditionally used to manage male sexual disorders including erectile dysfunction (ED. Methanol extract of BF (bark inhibited Rho-kinase 2 (ROCK-II enzyme activity in vitro with an IC50 of 20.29±1.83 μg/mL. The relaxant effect of methanol extract of BF (MEBF was studied on phenylephrine precontracted corpus cavernosum smooth muscle (CCSM isolated from young rats. The effect of MEBF treatment on sexual behaviour of both young (5 month and aged (24 month rats was also studied in addition to the influence on smooth muscle, collagen (collagen-I and -III level in penis, and sperm characteristics of young and aged rats. MEBF relaxed CCSM up to 21.77±2.57% and increased sexual behavior of young and aged rats. This increase in sexual function could be attributed to ROCK-II inhibition and increase in ratio of smooth muscle to collagen level in rat penile tissue. Increased sperm production and decreased defective sperms in young and aged rats corroborate the usefulness of Butea frondosa in male infertility in addition to ED.

  12. Chromatographic assay to study the activity of multiple enzymes involved in the synthesis and metabolism of dopamine and serotonin.

    Morgan, Lindsay D; Baker, Hannah; Yeoman, Mark S; Patel, Bhavik Anil

    2012-03-21

    Serotonin and dopamine are crucial regulators of signalling in the peripheral and central nervous systems. We present an ex-vivo, isocratic chromatographic method that allows for the measurement of tyrosine, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), tryptophan, 5-hydroxytryptophan (5-HTP), serotonin and 5-hydroxy-3-indoleacetic acid (5-HIAA) in a model central nervous (CNS) system, to study the role of key enzymes involved in the synthesis and metabolism of serotonin and dopamine. By utilising a sample splitting technique, we could test a single CNS sample at multiple time points under various pharmacological treatments. In, addition, we were able to conduct this assay by utilising the endogenous biochemical components of the CNS to study the synthesis and metabolism of serotonin and dopamine, negating the requirement of additional enzyme activators or stabilisers in the biological matrix. Finally we utilised NSD-1015, an aromatic amino acid decarboxylase enzyme inhibitor used to study the synthesis of dopamine and serotonin to monitor alterations in levels of key neurochemicals. 3-hydroxybenzylhydrazine dihydrochloride (NSD-1015) was able to reduce levels of serotonin and dopamine, whilst elevating precursors L-DOPA and 5-HTP. PMID:22290325

  13. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TRα1, TRβ1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TRα1, TRβ1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  14. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    Istrate, Monica A., E-mail: monicai@scripps.edu [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Nussler, Andreas K., E-mail: nuessler@uchir.me.tum.de [Department of Traumatology, Technical University Munich, Ismaningerstr. 22, 81675 Munich (Germany); Eichelbaum, Michel, E-mail: michel.eichelbaum@ikp-stuttgart.de [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Burk, Oliver, E-mail: oliver.burk@ikp-stuttgart.de [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany)

    2010-03-19

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  15. Right ventricular involvement in patients with Fabry's disease and the effect of enzyme replacement therapy

    According to echocardiography reports, Fabry cardiomyopathy not only affects the left ventricle (LV) but also the right ventricle (RV). Until now no MRI studies about the effect of enzyme replacement therapy (ERT) on the RV are available. We evaluated the effect of ERT on the RV. In this prospective trial 14 patients with genetically proven Fabry's disease were examined using a 1.5 T MR scanner before ERT and after 13 ± 1 months of ERT. All patients underwent cardiac MR imaging and the RV/LV cardiac morphology and function were analyzed. At baseline examination the values were as follows: RV mass 31 ± 6 g/m2, end-diastolic volume (EDV) 88 ± 13 ml/m2, end-systolic volume (ESV) 39 ± 9 ml/m2, stroke volume (SV) 49 ± 7 ml/m2 and ejection fraction (EF) 56 ± 5 %. The RV mass and EDV decreased significantly after 13 ± 1 months on ERT (mass 27 ± 7 g/m2, p 2, p 2), SV (43 ± 12 ml/m2) and EF (57 ± 7 %). The LV mass (102 ± 26 g/m2 vs. 94 ± 27 g/m2, p 2 vs. 66 ± 22 ml/m2, p 2 vs. 23 ± 9 ml/m2, p < 0.05) decreased significantly while the EF (64 ± 7 % vs. 66 ± 5 %; p < 0.05) increased significantly. Besides the known beneficial effect on the LV, ERT improves RV mass and EDV. (orig.)

  16. Cloning and expression of murine enzymes involved in the salvage pathway of GDP-L-fucose.

    Niittymäki, Jaana; Mattila, Pirkko; Roos, Christophe; Huopaniemi, Laura; Sjöblom, Solveig; Renkonen, Risto

    2004-01-01

    In the salvage pathway of GDP-L-fucose, free cytosolic fucose is phosphorylated by L-fucokinase to form L-fucose-L-phosphate, which is then further converted to GDP-L-fucose in the reaction catalyzed by GDP-L-fucose pyrophosphorylase. We report here the cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. Murine L-fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of L-fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP-L-fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP-L-fucose, the reaction product of GDP-L-pyrophosphorylase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine L-fucokinase and GDP-L-fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expression of L-fucokinase and GDP-L-fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a significant role in the synthesis of GDP-L-fucose in the cytosol. PMID:14686921

  17. Involvement of CYP 2E1 enzyme in ovotoxicity caused by 4-vinylcyclohexene and its metabolites

    4-Vinylcyclohexene (VCH) is bioactivated by hepatic CYP 2A and 2B to a monoepoxide (VCM) and subsequently to an ovotoxic diepoxide metabolite (VCD). Studies suggest that the ovary can directly bioactivate VCH via CYP 2E1. The current study was designed to evaluate the role of ovarian CYP 2E1 in VCM-induced ovotoxicity. Postnatal day 4 B6C3F1 and CYP 2E1 wild-type (+/+) and null (-/-) mouse ovaries were cultured (15 days) with VCD (30 μM), 1,2-VCM (125-1000 μM), or vehicle. Twenty-eight days female CYP 2E1 +/+ and -/- mice were dosed daily (15 days; ip) with VCH, 1,2-VCM, VCD or vehicle. Following culture or in vivo dosing, ovaries were histologically evaluated. In culture, VCD decreased (p 1 and CYP 2E1 +/+ ovaries, but not in CYP 2E1 -/- ovaries in culture. 1,2-VCM did not affect primary follicles in any group of mouse ovaries. Conversely, following in vivo dosing, primordial and primary follicles were reduced (p < 0.05) by VCD and VCM in CYP2E1 +/+ and -/-, and by VCH in +/+ mice. The data demonstrate that, whereas in vitro ovarian bioactivation of VCM requires CYP 2E1 enzyme, in vivo CYP 2E1 plays a minimal role. Thus, the findings support that hepatic metabolism dominates the contribution made by the ovary in bioactivation of VCM to its ovotoxic metabolite, VCD. This study also demonstrates the use of a novel ovarian culture system to evaluate ovary-specific metabolism of xenobiotics

  18. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    Liu W

    2015-10-01

    Full Text Available Wenqin Liu,1,2,* Jian Shi,1,2,* Lijun Zhu,2 Lingna Dong,1 Feifei Luo,2 Min Zhao,2 Ying Wang,2 Ming Hu,2,3 Linlin Lu,2 Zhongqiu Liu1,2 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China; 2International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China; 3Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, USA *These authors contributed equally to this work Abstract: Oxymatrine (OMT is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT, and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs and human intestinal microsomes (HIMs and the cytochrome P450 (CYP isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT

  19. Arsenite and its metabolites, MMAIII and DMAIII, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

    Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMAIII induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMAIII increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMAIII induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

  20. Effect of n-3 polyunsaturated fatty acid on gene expression of the critical enzymes involved in homocysteine metabolism

    Huang Tao

    2012-01-01

    Full Text Available Abstract Background Previous studies showed that plasma n-3 polyunsaturated fatty acid (PUFA was negatively associated with plasma homocysteine (Hcy. Objective We investigated the regulatory effect of n-3 PUFA on mRNA expression of the critical genes encoding the enzymes involved in Hcy metabolism. Methods HepG2 cells were treated with docosahexaenoic acid (DHA, eicosapentaenoic acid (EPA, alpha-linolenic acid (ALA respectively for 48 h. The cells were collected and total RNA was isolated. The mRNA expression levels of the genes were determined by using Real Time-PCR. Results Compared with controls, the mRNA expression levels of 5-methyltetrahydrofolate reductase (MTHFR were significantly increased in the DHA group (p Conclusions Our results suggest that DHA up-regulates CSE and MTHFR mRNA expression and down-regulates MAT mRNA expression involved in Hcy metabolism.

  1. The stress response of human proximal tubule cells to cadmium involves up-regulation of haemoxygenase 1 and metallothionein but not cytochrome P450 enzymes.

    Boonprasert, Kanyarat; Satarug, Soisungwan; Morais, Christudas; Gobe, Glenda C; Johnson, David W; Na-Bangchang, Kesara; Vesey, David A

    2016-05-13

    Enzymes of the cytochrome P450 (CYP) super-family are implicated in cadmium (Cd) -induced nephrotoxicity, however, direct evidence is lacking. This study investigated the endogenous expression of various CYP proteins together with the stress-response proteins, heme oxygenase-1 (HO-1) and metallothionein (MT) in human kidney sections and in cadmium-exposed primary cultures of human proximal tubular epithelial cells (PTC). By immunohistochemistry, the CYP members 2B6, 4A11 and 4F2 were prominently expressed in the cortical proximal tubular cells and to a lesser extent in distal tubular cells. Low levels of CYPs 2E1 and 3A4 were also detected. In PTC, in the absence of Cd, CYP2E1, CYP3A4, CYP4F2 and MT were expressed, but HO-1, CYP2B6 and CYP4A11 were not detected. A range of cadmium concentrations (0-100μM) were utilized to induce stress conditions. MT protein was further induced by as little as 0.5μM cadmium, reaching a 6-fold induction at 20μM, whereas for HO-1, a 5μM cadmium concentration was required for initial induction and at 20μM cadmium reached a 15-fold induction. The expression of CYP2E1, CYP3A4, and CYP4F2 were not altered by any cadmium concentrations tested at 48h. Cadmium caused a reduction in cell viability at concentrations above 10μM. In conclusion although cultured PTC, do express CYP proteins, (CYP2E1, CYP3A4, and CYP4F2), Cd-induced cell stress as indicted by induction of HO-1 and MT does not alter expression of these CYP proteins at 48h. PMID:27005776

  2. In situ analysis of enzymes involved in sucrose to hexose-phosphate conversion during stolon-to-tuber transition of potato

    Appeldoorn, N.J.G.; Sergeeva, L.; Vreugdenhil, D.; Plas, van der L.H.W.; Visser, R.G.F.

    2002-01-01

    An in situ study of enzymes involved in sucrose to hexose-phosphate conversion during in vitro stolon-to-tuber transition of potato (Solanum tuberosum L. cv. Bintje) was employed to follow developmental changes in spatial patterns. In situ activity of the respective enzymes was visualized by specifi

  3. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor.

    Kumagai, Takeshi; Aratsu, Yusuke; Sugawara, Ryosuke; Sasaki, Takamitsu; Miyairi, Shinichi; Nagata, Kiyoshi

    2016-04-01

    Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an anti-inflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-Lan-Gen, activated CYP3A4 gene transcription through the activation of the human PXR. PMID:26987505

  4. Screening of Chonemorpha fragrans Bioactive Extracts for Cytotoxicity Potential and Inhibition Studies of Key Enzymes Involved in Replication

    Kedari, Pradnya Prakash; Malpathak, Nutan Padmanabh

    2016-01-01

    Background: Chonemorpha fragrans (Moon) Alston, a liana belonging to family Apocynaceae, is used in traditional medicinal systems for the treatment of various ailments. It is an unexplored medicinal plant with respect to its anticancer potential. Objective: Cytotoxicity of sequential as well as crude extracts of in vivo plant parts (leaves, bark, and roots), in vitro cultures, and callus were compared. Materials and Methods: 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay was used to compare the extracts of various in vivo plant parts (leaves, bark, and roots) along with in vitro culture systems (in vitro plantlets, callus). Furthermore, the extracts were used to evaluate inhibition of key enzymes involved in replication, i.e. topoisomerase (Topo) I and II, DNA polymerase, to check the probable mechanism of action for this cytotoxicity. Results: MTT assay showed that the chloroform extract of callus has potent anticancer potential. The plant has a promising anticancer activity against human colon epithelium, lung carcinoma, and epidermoidal carcinoma cell lines. It was found to possess Topo as well as DNA polymerase inhibitory activity. Conclusion: The results have pointed toward pharmaceutical importance of this plant. This study is the first report of exploring the antiproliferative potential as well as inhibition studies of key enzymes involved in replication, which was useful to point out probable mechanism of action for extracts of C. fragrans. SUMMARY It's a first report of cytotoxicity studies and inhibition of enzyme involved in the replication process by Chonemorpha fragrans plant extracts. The results reveal the pharmaceutical importance of this plant. From various assays performed here, a potent anticancer potential of chloroform extract of callus was revealed showing Topo I (E. coli and human) inhibitory activity, DNA pol inhibitory activity. Considering the importance of these activities, plant further needs

  5. Evidence for a two-metal-ion mechanism in the cytidyltransferase KdsB, an enzyme involved in lipopolysaccharide biosynthesis.

    Helgo Schmidt

    Full Text Available Lipopolysaccharide (LPS is located on the surface of Gram-negative bacteria and is responsible for maintaining outer membrane stability, which is a prerequisite for cell survival. Furthermore, it represents an important barrier against hostile environmental factors such as antimicrobial peptides and the complement cascade during Gram-negative infections. The sugar 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo is an integral part of LPS and plays a key role in LPS functionality. Prior to its incorporation into the LPS molecule, Kdo has to be activated by the CMP-Kdo synthetase (CKS. Based on the presence of a single Mg²⁺ ion in the active site, detailed models of the reaction mechanism of CKS have been developed previously. Recently, a two-metal-ion hypothesis suggested the involvement of two Mg²⁺ ions in Kdo activation. To further investigate the mechanistic aspects of Kdo activation, we kinetically characterized the CKS from the hyperthermophilic organism Aquifex aeolicus. In addition, we determined the crystal structure of this enzyme at a resolution of 2.10 Å and provide evidence that two Mg²⁺ ions are part of the active site of the enzyme.

  6. Safety of Herbal Medicinal Products: Echinacea and Selected Alkylamides Do Not Induce CYP3A4 mRNA Expression

    Maryam Modarai

    2011-01-01

    Full Text Available A major safety concern with the use of herbal medicinal products (HMP is their interactions with conventional medicines, which are often mediated via the cytochrome P450 (CYP system. Echinacea is a widely used over-the-counter HMP, with proven immunomodulatory properties. Its increasing use makes research into its safety an urgent concern. Previously, we showed that Echinacea extracts and its alkylamides (thought to be important for Echinacea's immunomodulatory activity mildly inhibit the enzymatic activity of the main drug metabolising CYP isoforms, but to this date, there is insufficient work on its ability to alter CYP expression levels. We now report for the first time the effect of a commercial Echinacea extract (Echinaforce and four Echinacea alkylamides on the transcription of the major drug metabolizing enzyme CYP3A4. HepG2 cells were exposed for 96 h to clinically relevant concentrations of Echinaforce (22, 11.6 and 1.16 μg mL−1 or the alkylamides (1.62 and 44 nM. CYP3A4 mRNA levels were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR. Neither Echinaforce nor the alkylamides produced any significant changes in the steady-state CYP3A4 mRNA levels, under these conditions. In contrast, treatment with 50 μM rifampicin resulted in a 3.8-fold up-regulation over the vehicle control. We conclude that Echinaforce is unlikely to affect CYP3A4 transcriptional levels, even at concentrations which can inhibit the enzymatic activity of CYP3A4. Overall, our data provides further evidence for the lack of interactions between Echinacea and conventional drugs.

  7. Size and surface modification of amorphous silica particles determine their effects on the activity of human CYP3A4 in vitro

    Imai, Shunji; Yoshioka, Yasuo; Morishita, Yuki; Yoshida, Tokuyuki; Uji, Miyuki; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Higashisaka, Kazuma; Tsutsumi, Yasuo

    2014-12-01

    Because of their useful chemical and physical properties, nanomaterials are widely used around the world - for example, as additives in food and medicines - and such uses are expected to become more prevalent in the future. Therefore, collecting information about the effects of nanomaterials on metabolic enzymes is important. Here, we examined the effects of amorphous silica particles with various sizes and surface modifications on cytochrome P450 3A4 (CYP3A4) activity by means of two different in vitro assays. Silica nanoparticles with diameters of 30 and 70 nm (nSP30 and nSP70, respectively) tended to inhibit CYP3A4 activity in human liver microsomes (HLMs), but the inhibitory activity of both types of nanoparticles was decreased by carboxyl modification. In contrast, amine-modified nSP70 activated CYP3A4 activity. In HepG2 cells, nSP30 inhibited CYP3A4 activity more strongly than the larger silica particles did. Taken together, these results suggest that the size and surface characteristics of the silica particles determined their effects on CYP3A4 activity and that it may be possible to develop silica particles that do not have undesirable effects on metabolic enzymes by altering their size and surface characteristics.

  8. Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells.

    Negoro, Ryosuke; Takayama, Kazuo; Nagamoto, Yasuhito; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-04-15

    Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. PMID:26966071

  9. Involvement of cytochrome P-450 enzyme activity in the selectivity and safening action of pyrazosulfuron-ethyl.

    Yun, M S; Shim, I S; Usui, K

    2001-03-01

    To investigate the selectivity and safening action of the sulfonylurea herbicide pyrazosulfuron-ethyl (PSE), pyrazosulfuron-ethyl O-demethylase (PSEOD) activity involving oxidative metabolism by cytochrome P-450 was studied in rice (Oryza sativa L cv Nipponbare) and Cyperus serotinus Rottb. Cytochrome P-450-dependent activity was demonstrated by the use of the inducers 1,8-naphthalic anhydride and ethanol, the herbicides PSE, bensulfuron-methyl, dimepiperate and dymron, or the inhibitor piperonyl butoxide (PBO). Growth inhibition in C serotinus seedlings was more severe than that in rice seedlings. O-Dealkylation activities of PSE were induced differently in rice and in C serotinus, with distinctly higher activity in rice seedlings. The induced PSEOD activities were slightly inhibited by PBO in rice seedlings, whereas they were strongly inhibited in C serotinus seedlings. Dimepiperate and dymron were effective safeners of rice against PSE treatment. Treatments with herbicide alone resulted in less induction of PSEOD activity compared with combined treatments of the herbicide and safener. PSEOD activity in rice seedlings induced with herbicide alone was strongly inhibited by PBO, whereas it was weakly inhibited in rice seedlings induced with combinations of PSE and two safeners. These results suggest that O-demethylation by cytochrome P-450 enzymes may be involved in the metabolism of PSE and may contribute to its selectivity and safening action. Furthermore, these results suggest the existence of a multiple form of cytochrome P-450 in plants. PMID:11455659

  10. In vitro inhibition of CYP3A4 by herbal remedies frequently used by cancer patients.

    Engdal, Silje; Nilsen, Odd Georg

    2009-07-01

    The herbal remedies Natto K2, Agaricus, mistletoe, noni juice, green tea and garlic, frequently used by cancer patients, were investigated for their in vitro inhibition potential of cytochrome P-450 3A4 (CYP3A4) metabolism. To our knowledge, only garlic and green tea had available data on the possible inhibition of CYP3A4 metabolism. Metabolic studies were performed with human c-DNA baculovirus expressed CYP3A4. Testosterone was used as a substrate and ketoconazole as a positive quantitative inhibition control. The formation of 6-beta-OH-testosterone was quantified by a validated HPLC methodology. Green tea was the most potent inhibitor of CYP3A4 metabolism (IC(50): 73 microg/mL), followed by Agaricus, mistletoe and noni juice (1324, 3594, >10 000 microg/mL, respectively). All IC(50) values were high compared with those determined for crude extracts of other herbal remedies. The IC(50)/IC(25) ratios for the inhibiting herbal remedies ranged from 2.15 to 2.67, indicating similar inhibition profiles of the herbal inhibitors of CYP3A4. Garlic and Natto K2 were classified as non-inhibitors. Although Agaricus, noni juice, mistletoe and green tea inhibited CYP3A4 metabolism in vitro, clinically relevant systemic or intestinal interactions with CYP3A4 were considered unlikely, except for a probable inhibition of intestinal CYP3A4 by the green tea product. PMID:19170155

  11. H2O2-Activated Up-Regulation of Glutathione in Arabidopsis Involves Induction of Genes Encoding Enzymes Involved in Cysteine Synthesis in the Chloroplast

    Guillaume Queval; Dorothée Thominet; Hélène Vanacker; Myroslawa Miginiac-Maslow; Bertrand Gakière; Graham Noctor

    2009-01-01

    Glutathione is a key player in cellular redox homeostasis and, therefore, in the response to H2O2, but the factors regulating oxidation-activated glutathione synthesis are still unclear. We investigated H2O2-induced glutathione synthesis in a conditional Arabidopsis catalase-deficient mutant (cat2). Plants were grown from seed at elevated CO2 for 5 weeks, then transferred to air in either short-day or long-day conditions. Compared to cat2 at elevated CO2 or wild-type plants in any condition, transfer of cat2 to air in both photoperiods caused measurable oxidation of the leaf glutathione pool within hours. Oxidation continued on subsequent days and was accompanied by accumulation of glutathione. This effect was stronger in cat2 transferred to air in short days, and was not linked to appreciable increases in the extractable activities of or transcripts encoding enzymes involved in the committed pathway of glutathione synthesis. In contrast, it was accompanied by increases in serine, O-acetylserine, and cysteine. These changes in metabolites were accompanied by induction of genes encoding adenosine phosphosulfate reductase (APR), particularly APR3, as well as a specific serine acetyltransferase gene (SAT2.1) encoding a chloroplastic SAT. Marked induction of these genes was only observed in cat2 transferred to air in short-day conditions, where cysteine and glutathione accumulation was most dramatic. Unlike other SAT genes, which showed negligible induction in cat2, the relative abundance of APR and SAT2.1 transcripts was closely correlated with marker transcripts for H2O2 signaling. Together, the data underline the importance of cysteine synthesis in oxidant-induced up-regulation of glutathione synthesis and suggest that the chloroplast makes an important contribution to cysteine production under these circumstances.

  12. Aluminum-induced decrease in CO{sub 2} assimilation in citrus seedlings is accompanied by decreased activities of key enzymes involved in CO{sub 2} assimilation

    Chen, L-S.; Liu, X-H. [Fujian Agriculture and Forestry University, College of Horticulture, Fuzhou (China); Qi, Y-P. [Fujian Provincial Institute of Medical Sciences, Fuzhou (China); Smith, B. R. [Cornell University, Dept. of Horticulture, Ithaca, NY (United States)

    2005-03-01

    Many previous studies provided evidence that heavy metals affect the carbon dioxide assimilation of plants by inhibiting different enzymes involved in the Calvin cycle. Other studies have shown little or no change in the activities of Calvion cycle enzymes accompanying a heavy metal-induced decline in carbon dioxide assimilation. This study reexamined aluminium toxicity in leaf carbohydrate metabolism by determining responses in citrus leaves to aluminium, specifically in terms of carbon dioxide assimilation, key enzymes in the Calvin cycle, starch and sucrose syntheses, non-structural carbohydrates, and photosynthetic intermediates. Based on results in each of these areas, it was concluded that aluminium decreases carbon dioxide assimilation, but in so doing either increases or has no effect on the activities of enzymes involved in the Calvin cycle. The decrease in carbon dioxide assimilation induced by aluminium is suspected to be the result of increased photorespiration. 41 refs., 5 tabs., 2 figs.

  13. In vitro characterization of the enzymes involved in TDP-D-forosamine biosynthesis in the spinosyn pathway of Saccharopolyspora spinosa.

    Hong, Lin; Zhao, Zongbao; Melançon, Charles E; Zhang, Hua; Liu, Hung-wen

    2008-04-01

    Forosamine (4-dimethylamino)-2,3,4,6-tetradeoxy-beta-D-threo-hexopyranose) is a highly deoxygenated sugar component of several important natural products, including the potent yet environmentally benign insecticide spinosyns. To study D-forosamine biosynthesis, the five genes (spnO, N, Q, R, and S) from the spinosyn gene cluster thought to be involved in the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-D-forosamine were cloned and heterologously expressed, and the corresponding proteins were purified and their activities examined in vitro. Previous work demonstrated that SpnQ functions as a pyridoxamine 5'-monophosphate (PMP)-dependent 3-dehydrase which, in the presence of the cellular reductase pairs ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase, catalyzes C-3 deoxygenation of TDP-4-keto-2,6-dideoxy-D-glucose. It was also established that SpnR functions as a transaminase which converts the SpnQ product, TDP-4-keto-2,3,6-trideoxy-D-glucose, to TDP-4-amino-2,3,4,6-tetradeoxy-D-glucose. The results presented here provide a full account of the characterization of SpnR and SpnQ and reveal that SpnO and SpnN functions as a 2,3-dehydrase and a 3-ketoreductase, respectively. These two enzymes act sequentially to catalyze C-2 deoxygenation of TDP-4-keto-6-deoxy-D-glucose to form the SpnQ substrate, TDP-4-keto-2,6-dideoxy-D-glucose. Evidence has also been obtained to show that SpnS functions as the 4-dimethyltransferase that converts the SpnR product to TDP-D-forosamine. Thus, the biochemical functions of the five enzymes involved in TDP-D-forosamine formation have now been fully elucidated. The steady-state kinetic parameters for the SpnQ-catalyzed reaction have been determined, and the substrate specificities of SpnQ and SpnR have been explored. The implications of this work for natural product glycodiversification and comparative mechanistic analysis of SpnQ and related NDP-sugar 3-dehydrases E1 and ColD are discussed. PMID:18345667

  14. CYP3A4 mediated in vitro metabolism of vinflunine in human liver microsomes

    Xiao-ping ZHAO; Jiao ZHONG; Xiao-quan LIU; Guang-ji WANG

    2007-01-01

    Aim: To study the metabolism of vinflunine and the effects of selective cyto-chrome P-450 (CYP450) inhibitors on the metabolism of vinflunine in human liver microsomes. Methods: Individual selective CYP450 inhibitors were used to inves-tigate their effects on the metabolism of vinflunine and the principal CYP450 isoform involved in the formation of metabolites M1 and M2 in human liver microsomes.Results: Vinflunine was rapidly metabolized to 2 metabolites: M1 and M2 in human liver microsomes. M1 and M2 were tentatively presumed to be the N-oxide metabo-lite or hydroxylated metabolite and epoxide metabolite of vinflunine, respectively. Ketoconazole uncompetitively inhibited the formation of M1, and competitively inhibited the formation of M2, while α-naphthoflavone, sulfaphenazole, diethyl dithiocarbamate, tranylcypromine and quinidine had little or no inhibitory effect on the formation of M1 and M2. Conclusion: Vinflunine is rapidly metabolized in human liver microsomes, and CYP3A4 is the major human CYP450 involved in the metabolism of vinflunine.

  15. Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus

    Tahlan, Kapil; Park, Hyeon Ung; Wong, Annie; Beatty, Perrin H.; Jensen, Susan E.

    2004-01-01

    Recently, a second copy of a gene encoding proclavaminate amidinohydrolase (pah1), an enzyme involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in Streptomyces clavuligerus, was identified and isolated. Using Southern analysis, we have now isolated second copies of the genes encoding the carboxyethylarginine synthase (ceaS) and β-lactam synthetase (bls) enzymes. These new paralogues are given the gene designations ceaS1 and bls1 and are located immediately upst...

  16. Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

    2008-01-01

    The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.

  17. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 μM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 μM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 μM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 μM and 10 μM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

  18. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

    Casabar, Richard C T; Das, Parikshit C; Dekrey, Gregory K; Gardiner, Catherine S; Cao, Yan; Rose, Randy L; Wallace, Andrew D

    2010-06-15

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates. PMID:20361990

  19. Molecular modeling and simulation of FabG, an enzyme involved in the fatty acid pathway of Streptococcus pyogenes.

    Shafreen, Rajamohmed Beema; Pandian, Shunmugiah Karutha

    2013-09-01

    Streptococcus pyogenes (SP) is the major cause of pharyngitis accompanied by strep throat infections in humans. 3-keto acyl reductase (FabG), an important enzyme involved in the elongation cycle of the fatty acid pathway of S. pyogenes, is essential for synthesis of the cell-membrane, virulence factors and quorum sensing-related mechanisms. Targeting SPFabG may provide an important aid for the development of drugs against S. pyogenes. However, the absence of a crystal structure for FabG of S. pyogenes limits the development of structure-based drug designs. Hence, in the present study, a homology model of FabG was generated using the X-ray crystallographic structure of Aquifex aeolicus (PDB ID: 2PNF). The modeled structure was refined using energy minimization. Furthermore, active sites were predicted, and a large dataset of compounds was screened against SPFabG. The ligands were docked using the LigandFit module that is available from Discovery Studio version 2.5. From this list, 13 best hit ligands were chosen based on the docking score and binding energy. All of the 13 ligands were screened for Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. From this, the two best descriptors, along with one descriptor that lay outside the ADMET plot, were selected for molecular dynamic (MD) simulation. In vitro testing of the ligands using biological assays further substantiated the efficacy of the ligands that were screened based on the in silico methods. PMID:23988477

  20. Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis.

    Wang, Hai-Yan; Zhang, Ji; Zhang, Yue-Jing; Zhang, Bo; Liu, Chong-Xi; He, Hai-Rong; Wang, Xiang-Jing; Xiang, Wen-Sheng

    2014-12-01

    Milbemycin oxime has been commercialized as effective anthelmintics in the fields of animal health, agriculture, and human infections. Currently, milbemycin oxime is synthesized by a two-step chemical reaction, which involves the ketonization of milbemycins A3/A4 to yield the intermediates 5-oxomilbemycins A3/A4 using CrO3 as catalyst. Due to the low efficiency and environmental unfriendliness of the ketonization of milbemycins A3/A4, it is imperative to develop alternative strategies to produce 5-oxomilbemycins A3/A4. In this study, the atmospheric and room temperature plasma (ARTP) mutation system was first employed to treat milbemycin-producing strain Streptomyces bingchenggensis, and a mutant strain BC-120-4 producing milbemycins A3, A4, B2, and B3 as main components was obtained, which favors the construction of genetically engineered strains producing 5-oxomilbemycins. Importantly, the milbemycins A3/A4 yield of BC-120-4 reached 3,890 ± 52 g/l, which was approximately two times higher than that of the initial strain BC-109-6 (1,326 ± 37 g/l). The subsequent interruption of the gene milF encoding a C5-ketoreductase responsible for the ketonization of milbemycins led to strain BCJ60 (∆milF) with the production of 5-oxomilbemycins A3/A4 and the elimination of milbemycins A3, A4, B2, and B3. The high 5-oxomilbemycins A3/A4 yield (3,470 ± 147 g/l) and genetic stability of BCJ60 implied the potential use in industry to prepare 5-oxomilbemycins A3/A4 for the semisynthesis of milbemycins oxime. PMID:25081559

  1. Recent insight on the control of enzymes involved in estrogen formation and transformation in human breast cancer.

    Pasqualini, Jorge R; Chetrite, Gérard S

    2005-02-01

    The great majority of breast cancers are in their early stage hormone-dependent and it is well accepted that estradiol (E2) plays an important role in the genesis and evolution of this tumor. Human breast cancer tissues contain all the enzymes: estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase involved in the last steps of E2 bioformation. Sulfotransferases which convert estrogens into the biologically inactive estrogen sulfates are also present in this tissue. Quantitative data show that the 'sulfatase pathway', which transforms estrogen sulfates into the bioactive unconjugated E2, is 100-500 times higher than the 'aromatase pathway', which converts androgens into estrogens. The treatment of breast cancer patients with anti-aromatases is largely developed with very positive results. However, the formation of E2 via the 'sulfatase pathway' is very important in the breast cancer tissue. In recent years it was found that antiestrogens (e.g. tamoxifen, 4-hydroxytamoxifen), various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, dydrogesterone, norelgestromin), tibolone and its metabolites, as well as other steroidal (e.g. sulfamates) and non-steroidal compounds, are potent sulfatase inhibitors. In another series of studies, it was found that E2 itself has a strong anti-sulfatase action. This paradoxical effect of E2 adds a new biological response of this hormone and could be related to estrogen replacement therapy in which it was observed to have either no effect or to decrease breast cancer mortality in postmenopausal women. Interesting information is that high expression of steroid sulfatase mRNA predicts a poor prognosis in patients with +ER. These progestins, as well as tibolone, can also block the conversion of estrone to estradiol by the inhibition of the 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD-1). High expressison of 17beta-HSD-1 can be an indicator of adverse prognosis in ER-positive patients. It was shown that

  2. Cytochrome P450 3A4FNx011B as pharmacogenomic predictor of tacrolimus pharmacokinetics and clinical outcome in the liver transplant recipients

    Abdulkareem Albekairy

    2013-01-01

    Full Text Available Background and Aims: Tacrolimus is a macrolide immunosuppressant used for prevention of allograft rejection in organ transplantation and metabolized in the liver and intestine by cytochrome P450 3A4 (CYP3A4 enzyme. A single nucleotide polymorphism (SNP in the CYP3A4 promoter region has been identified. It has been shown that the presence of CYP3A4FNx011B allele (variant GG is associated with a reduced catalytic activity of CYP3A4 in vivo. The aim of this study was to determine the role of CYP3A4FNx011B on tacrolimus dosing and clinical outcome in liver transplant recipients. Subjects and Methods: Forty-eight liver transplant recipients were stratified according to the genotype. There were 32 wild-type (AA patients and 5 homozygous variant (GG and 11 (AG heterozygous. Tacrolimus doses and trough concentrations as well as phenotypic data were collected in the first 10 days of the transplant. Results: The tacrolimus concentration was significantly higher in the wild (AA group as compared to homozygous variant (GG and heterozygous (AG patients. Homozygous variant (GG group had significantly lower dose requirements. However, no significant difference was observed in the concentration/dose ratio between all groups. Conclusions: Based on our results, it may be concluded that CYP3A4FNx011B of recipient is an important factor influencing pharmacokinetic of tacrolimus, as patients with CYP3A4FNx011B polymorphism may require lower tacrolimus doses to maintain therapeutic levels. The dose reduction may not affect clinical outcomes after liver transplant.

  3. Expression of membrane transporters and metabolic enzymes involved in estrone-3-sulphate disposition in human breast tumour tissues.

    Banerjee, Nilasha; Miller, Naomi; Allen, Christine; Bendayan, Reina

    2014-06-01

    Two-thirds of newly diagnosed hormone-dependent (HR?) breast cancers are detected in post-menopausal patients where estrone-3-sulphate (E3S) is the predominant source for tumour estradiol. Understanding intra-tumoral fate of E3S would facilitate in the identification of novel molecular targets for HR? post-menopausal breast cancer patients. Hence this study investigates the clinical expression of (i) organic anion-transporting polypeptides (OATPs), (ii) multidrug resistance protein (MRP-1), breast cancer resistance proteins (BCRP), and (iii) sulphatase (STS), 17β-hydroxysteroid dehydrogenase (17β-HSD-1), involved in E3S uptake, efflux and metabolism, respectively. Fluorescent and brightfield images of stained tumour sections (n = 40) were acquired at 4× and 20× magnification, respectively. Marker densities were measured as the total area of positive signal divided by the surface area of the tumour section analysed and was reported as % area (ImageJ software). Tumour, stroma and non-tumour tissue areas were also quantified (Inform software), and the ratio of optical intensity per histologic area was reported as % area/tumour, % area/stroma and % area/non-tumour. Functional role of OATPs and STS was further investigated in HR? (MCF-7, T47-D, ZR-75) and HR-(MDA-MB-231) cells by transport studies conducted in the presence or absence of specific inhibitors. Amongst all the transporters and enzymes, OATPs and STS have significantly (p < 0.0001) higher expression in HR? tumour sections with highest target signals obtained from the tumour regions of the tissues. Specific OATP-mediated E3S uptake and STS-mediated metabolism were also observed in all HR? breast cancer cells. These observations suggest the potential of OATPs as novel molecular targets for HR? breast cancers. PMID:24831777

  4. Key Feature of the Catalytic Cycle of TNF-α Converting Enzyme Involves Communication Between Distal Protein Sites and the Enzyme Catalytic Core

    Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal-protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-α converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place between distal protein sites and the enzyme catalytic core. The observed charge transitions are synchronized with distinct phases in the reaction kinetics and changes in metal coordination chemistry mediated by the binding of the peptide substrate to the catalytic metal ion and product release. Here we report key local charge transitions critical for proteolysis as well as long sought evidence for the proposed reaction model of peptide hydrolysis. This study provides a general approach for gaining critical insights into the molecular basis of substrate recognition and turnover by zinc metalloproteinases that may be used for drug design

  5. Linkage and association of haplotypes at the APOA1/C3/A4/A5 genecluster to familial combined hyperlipidemia

    Eichenbaum-Voline, Sophie; Olivier, Michael; Jones, Emma L.; Naoumova, Rossitza P.; Jones, Bethan; Gau, Brian; Seed, Mary; Betteridge,D. John; Galton, David J.; Rubin, Edward M.; Scott, James; Shoulders,Carol C.; Pennacchio, Len A.

    2002-09-15

    Combined hyperlipidemia (CHL) is a common disorder of lipidmetabolism that leads to an increased risk of cardiovascular disease. Thelipid profile of CHL is characterised by high levels of atherogeniclipoproteins and low levels of high-density-lipoprotein-cholesterol.Apolipoprotein (APO) A5 is a newly discovered gene involved in lipidmetabolism located within 30kbp of the APOA1/C3/A4 gene cluster. Previousstudies have indicated that sequence variants in this cluster areassociated with increased plasma lipid levels. To establish whethervariation at the APOA5 gene contributes to the transmission of CHL, weperformed linkage and linkage disequilibrium (LD) tests on a large cohortof families (n=128) with familial CHL (FCHL). The linkage data producedevidence for linkage of the APOA1/C3/A4/A5 genomic interval to FCHL (NPL= 1.7, P = 0.042). The LD studies substantiated these data. Twoindependent rare alleles, APOA5c.56G and APOC3c.386G of this gene clusterwere over-transmitted in FCHL (P = 0.004 and 0.007, respectively), andthis was associated with a reduced transmission of the most commonAPOA1/C3/A4/A5 haplotype (frequency 0.4425) to affected subjects (P =0.013). The APOA5c.56G allele was associated with increased plasmatriglyceride levels in FCHL probands, whereas the second, andindependent, APOC3c.386G allele was associated with increased plasmatriglyceride levels in FCHL pedigree founders. Thus, this allele (or anallele in LD) may mark a quantitative trait associated with FCHL, as wellas representing a disease susceptibility locus for the condition. Thisstudy establishes that sequence variation in the APOA1/C3/A4/A5 genecluster contributes to the transmission of FCHL in a substantialproportion of affected families, and that these sequence variants mayalso contribute to the lipid abnormalities of the metabolic syndrome,which is present in up to 40 percent of persons with cardiovasculardisease.

  6. Enzymes Involved in the Aerobic Bacterial Degradation of N-Heteroaromatic Compounds: Molybdenum Hydroxylases and Ring-Opening 2,4-Dioxygenases

    Fetzner, S.

    Many N-heteroaromatic compounds are utilized by micro-organisms as a source of carbon (and nitrogen) and energy. The aerobic bacterial degradation of these growth substrates frequently involves several hydroxylation steps and subsequent dioxygenolytic cleavage of (di)hydroxy-substituted heteroaromatic intermediates to aliphatic metabolites which finally are channeled into central metabolic pathways. As a rule, the initial bacterial hydroxylation of a N-heteroaromatic compound is catalyzed by a molybdenum hydroxylase, which uses a water molecule as source of the incorporated oxygen. The enzyme's redox-active centers - the active site molybdenum ion coordinated to a distinct pyranopterin cofactor, two different [2Fe2S] centers, and in most cases, flavin adenine dinucleotide - transfer electrons from the N-heterocyclic substrate to an electron acceptor, which for many molybdenum hydroxylases is still unknown. Ring-opening 2,4-dioxygenases involved in the bacterial degradation of quinaldine and 1H-4-oxoquinoline catalyze the cleavage of two carbon-carbon bonds with concomitant formation of carbon monoxide. Since they contain neither a metal center nor an organic cofactor, and since they do not show any sequence similarity to known oxygenases, these unique dioxygenases form a separate enzyme family. Quite surprisingly, however, they appear to be structurally and mechanistically related to enzymes of the α/β hydrolase fold superfamily. Microbial enzymes are a great resource for biotechnological applications. Microbial strains or their enzymes may be used for degradative (bioremediation) or synthetic (biotransformation) purposes. Modern bioremediation or biotransformation strategies may even involve microbial catalysts or strains designed by protein engineering or pathway engineering. Prerequisite for developing such modern tools of biotechnology is a comprehensive understanding of microbial metabolic pathways, of the structure and function of enzymes, and of the

  7. Up-regulating CYP3A4 expression in C3A cells by transfection with a novel chimeric regulator of hPXR-p53-AD.

    Feng Chen

    Full Text Available Most hepatoma cell lines lack proper expression and induction of CYP3A4 enzyme, which limits their use for predicting drug metabolism and toxicity. Nuclear receptor pregnane X receptor (PXR has been well recognized for its critical role in regulating expression of CYP3A4 gene. However, its physiological activity of binding to the particular site of promoter is significantly weakened in hepatic cell lines. To address this problem, we created "chimeric PXR" constructs by appending a strong activation domain (AD from p53 subunit to either N- or C- termini of the human PXR (hPXR, that is, hPXR-p53 and p53-hPXR. C3A, a hepatoma cell line, was used as the cell model to test the regulation effect of chimeric hPXR over wild type (WT hPXR on CYP3A4 expression at gene, protein, and metabolism levels, respectively. Compared with C3A cells transiently transfected with WT hPXR, the activity of CYP3A4.XREM.luc reporter gene in C3A cells transfected with hPXR-p53 or p53-hPXR increased 5- and 9-fold respectively, and the levels of CYP3A4 mRNA expression increased 3.5- and 2.6-fold, respectively. C3A cells stably transfected with hPXR-p53-AD exhibited an improved expression of CYP3A4 at both gene (2-fold and protein (1.5-fold levels compared to WT C3A cells. Testosterone, a CYP3A4-specific substrate, was used for detecting the metabolism activity of CYP3A4. No testosterone metabolite could be detected in microsomes from WT C3A cells and WT C3A cells-based array, while the formation of 6β-hydroxytestosterone metabolite in the transfected cells was 714 and 55 pmol/mg protein/min, respectively. In addition, all the above expression levels in the transfected cell models could be further induced with additional treatment of Rifampicin, a specific inducer for CYP3A4. In conclusion, our study established a proof-of-principle example that genetic modification with chimeric hPXR-p53-AD could improve CYP3A4 metabolism ability in hepatic cell line.

  8. Pi-pi Stacking Mediated Cooperative Mechanism for Human Cytochrome P450 3A4

    Botao Fa

    2015-04-01

    Full Text Available Human Cytochrome P450 3A4 (CYP3A4 is an important member of the cytochrome P450 superfamily with responsibility for metabolizing ~50% of clinical drugs. Experimental evidence showed that CYP3A4 can adopt multiple substrates in its active site to form a cooperative binding model, accelerating substrate metabolism efficiency. In the current study, we constructed both normal and cooperative binding models of human CYP3A4 with antifungal drug ketoconazoles (KLN. Molecular dynamics simulation and free energy calculation were then carried out to study the cooperative binding mechanism. Our simulation showed that the second KLN in the cooperative binding model had a positive impact on the first one binding in the active site by two significant pi-pi stacking interactions. The first one was formed by Phe215, functioning to position the first KLN in a favorable orientation in the active site for further metabolism reactions. The second one was contributed by Phe304. This pi-pi stacking was enhanced in the cooperative binding model by the parallel conformation between the aromatic rings in Phe304 and the dioxolan moiety of the first KLN. These findings can provide an atomic insight into the cooperative binding in CYP3A4, revealing a novel pi-pi stacking mechanism for drug-drug interactions.

  9. High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in Nicotiana tabacum

    Synková, Helena; Semorádová, Šárka; Burketová, Lenka

    2004-01-01

    Roč. 79, č. 2 (2004), s. 169-179. ISSN 0167-6857 R&D Projects: GA ČR GA206/01/1061; GA ČR GA206/03/0310 Grant ostatní: Grantová agentura Univerzity Karlovy(CZ) 134/2001/B-Bio/PrF; Grantová agentura Univerzity Karlovy(CZ) Z5038910 Institutional research plan: CEZ:AV0Z5038910 Keywords : antioxidant enzymes * enzymes of intermediary metabolism * ex vitro Subject RIV: ED - Physiology Impact factor: 1.028, year: 2004

  10. Lipolytic enzymes involving lipolysis in Teleost: Synteny, structure, tissue distribution, and expression in grass carp (Ctenopharyngodon idella).

    Sun, Jian; Ji, Hong; Li, Xue-Xian; Shi, Xiao-Chen; Du, Zhen-Yu; Chen, Li-Qiao

    2016-08-01

    Lipolysis is the biochemical pathway responsible for the sequential hydrolysis of triacylglycerols (TAGs) stored in cellular lipid droplets. Three enzymes are known to participate in TAGs hydrolysis, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase (MGL), and each is present in mammals as only one isoform. Here we show that the genome of grass carp (Ctenopharyngodon idella) and other teleosts codes for one ATGL, two HSLs, and one MGL isoforms. Two isoforms of HSL gene, HSLa and HSLb, derived from paralogous genes that could be originated from teleost-specific genome duplication (TSGD) event. The genes encoding for fish ATGL and MGL were conserved and contained nine and seven coding exons, respectively. However, two isoforms of HSL gene had a remarkable variation in gene structure, such as HSLa gene contained ten and HSLb contained thirteen exons. All three enzymes, including two isoforms of HSL, were expressed in a wide range of tissues, but the abundance of each gene mRNA showed the tissue-dependent expression patterns. During fasting, only ATGL and HSLa showed a significant increase in adipose tissue and adipocyte, indicating that ATGL and HSLa may be the main rate-limiting enzymes controlling the hydrolysis of TAGs in fasting-induced lipolysis. Different expression of HSLa and HSLb suggests that they might serve different roles in fasting-induced lipolysis. These results provide evidence about the conservation and divergence of genes of fish lipolytic enzymes. PMID:27131420

  11. Going Beyond Common Drug Metabolizing Enzymes: Case Studies of Biotransformation Involving Aldehyde Oxidase, γ-Glutamyl Transpeptidase, Cathepsin B, Flavin-Containing Monooxygenase, and ADP-Ribosyltransferase.

    Fan, Peter W; Zhang, Donglu; Halladay, Jason S; Driscoll, James P; Khojasteh, S Cyrus

    2016-08-01

    The significant roles that cytochrome P450 (P450) and UDP-glucuronosyl transferase (UGT) enzymes play in drug discovery cannot be ignored, and these enzyme systems are commonly examined during drug optimization using liver microsomes or hepatocytes. At the same time, other drug-metabolizing enzymes have a role in the metabolism of drugs and can lead to challenges in drug optimization that could be mitigated if the contributions of these enzymes were better understood. We present examples (mostly from Genentech) of five different non-P450 and non-UGT enzymes that contribute to the metabolic clearance or bioactivation of drugs and drug candidates. Aldehyde oxidase mediates a unique amide hydrolysis of GDC-0834 (N-[3-[6-[4-[(2R)-1,4-dimethyl-3-oxopiperazin-2-yl]anilino]-4-methyl-5-oxopyrazin-2-yl]-2-methylphenyl]-4,5,6,7-tetrahydro-1-benzothiophene-2-carboxamide), leading to high clearance of the drug. Likewise, the rodent-specific ribose conjugation by ADP-ribosyltransferase leads to high clearance of an interleukin-2-inducible T-cell kinase inhibitor. Metabolic reactions by flavin-containing monooxygenases (FMO) are easily mistaken for P450-mediated metabolism such as oxidative defluorination of 4-fluoro-N-methylaniline by FMO. Gamma-glutamyl transpeptidase is involved in the initial hydrolysis of glutathione metabolites, leading to formation of proximate toxins and nephrotoxicity, as is observed with cisplatin in the clinic, or renal toxicity, as is observed with efavirenz in rodents. Finally, cathepsin B is a lysosomal enzyme that is highly expressed in human tumors and has been targeted to release potent cytotoxins, as in the case of brentuximab vedotin. These examples of non-P450- and non-UGT-mediated metabolism show that a more complete understanding of drug metabolizing enzymes allows for better insight into the fate of drugs and improved design strategies of molecules in drug discovery. PMID:27117704

  12. GW4064, an Agonist of Farnesoid X Receptor, Represses CYP3A4 Expression in Human Hepatocytes by Inducing Small Heterodimer Partner Expression

    Zhang, Shu; Pan, Xian; Jeong, Hyunyoung

    2015-01-01

    Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis and is recognized as a promising therapeutic target for metabolic diseases. The biologic function of FXR is mediated in part by a small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, and SHP in turn represses the activity of multiple transcription factors. This study aimed to investigate the effect of FXR activation on expression of the major drug-metabolizing enzyme CYP3A4. The ef...

  13. Paramecium bursaria Chlorella virus 1 encodes two enzymes involved in the biosynthesis of GDP-L-fucose and GDP-D-rhamnose.

    Tonetti, Michela; Zanardi, Davide; Gurnon, James R; Fruscione, Floriana; Armirotti, Andrea; Damonte, Gianluca; Sturla, Laura; De Flora, Antonio; Van Etten, James L

    2003-06-13

    At least three structural proteins in Paramecium bursaria Chlorella virus (PBCV-1) are glycosylated, including the major capsid protein Vp54. However, unlike other glycoprotein-containing viruses that use host-encoded enzymes in the endoplasmic reticulum-Golgi to glycosylate their proteins, PBCV-1 encodes at least many, if not all, of the glycosyltransferases used to glycosylate its structural proteins. As described here, PBCV-1 also encodes two open reading frames that resemble bacterial and mammalian enzymes involved in de novo GDP-L-fucose biosynthesis. This pathway, starting from GDP-D-mannose, consists of two sequential steps catalyzed by GDP-D-mannose 4,6 dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, respectively. The two PBCV-1-encoded genes were expressed in Escherichia coli, and the recombinant proteins had the predicted enzyme activity. However, in addition to the dehydratase activity, PBCV-1 GMD also had a reductase activity, producing GDP-D-rhamnose. In vivo studies established that PBCV-1 GMD and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are expressed after virus infection and that both GDP-L-fucose and GDP-D-rhamnose are produced in virus-infected cells. Thus, PBCV-1 is the first virus known to encode enzymes involved in nucleotide sugar metabolism. Because fucose and rhamnose are components of the glycans attached to Vp54, the pathway could circumvent a limited supply of GDP sugars by the algal host. PMID:12679342

  14. Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

    Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222. Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å

  15. Vitamin K2 in Electron Transport System: Are Enzymes Involved in Vitamin K2 Biosynthesis Promising Drug Targets?

    Eeshwaraiah Begari; Michio Kurosu

    2010-01-01

    Aerobic and anaerobic respiratory systemsallow cells to transport the electrons to terminal electron acceptors. The quinone (ubiquinone or menaquinone) pool is central to the electron transport chain. In the majority of Gram-positive bacteria, vitamin K2 (menaquinone) is the sole quinone in the electron transport chain, and thus, the bacterial enzymes catalyzing the synthesis of menaquinone are potential targets for the development of novel antibacterial drugs. This manuscript reviews the rol...

  16. A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation

    Cazzonelli, Christopher I.; Nisar, Nazia; Roberts, Andrea C.; Murray, Kevin D.; Borevitz, Justin O; Pogson, Barry J.

    2014-01-01

    Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzym...

  17. Unique behavior of Trypanosoma cruzi mevalonate kinase: A conserved glycosomal enzyme involved in host cell invasion and signaling.

    Ferreira, Éden Ramalho; Horjales, Eduardo; Bonfim-Melo, Alexis; Cortez, Cristian; da Silva, Claudio Vieira; De Groote, Michel; Sobreira, Tiago José Paschoal; Cruz, Mário Costa; Lima, Fabio Mitsuo; Cordero, Esteban Mauricio; Yoshida, Nobuko; da Silveira, José Franco; Mortara, Renato Arruda; Bahia, Diana

    2016-01-01

    Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion. PMID:27113535

  18. Unique behavior of Trypanosoma cruzi mevalonate kinase: A conserved glycosomal enzyme involved in host cell invasion and signaling

    Ferreira, Éden Ramalho; Horjales, Eduardo; Bonfim-Melo, Alexis; Cortez, Cristian; da Silva, Claudio Vieira; De Groote, Michel; Sobreira, Tiago José Paschoal; Cruz, Mário Costa; Lima, Fabio Mitsuo; Cordero, Esteban Mauricio; Yoshida, Nobuko; da Silveira, José Franco; Mortara, Renato Arruda; Bahia, Diana

    2016-01-01

    Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion. PMID:27113535

  19. Global (Q)SAR models on substrates for human Cytochrome P450 3A4

    Ringsted, Tine; Nikolov, Nikolai Georgiev; Wedebye, Eva Bay;

    prescribed drugs. Literature on substrates and non-substrates primarily based on in vivo human data on the CYP 3A4 isoenzyme was collected and a training set of 863 chemicals was used to create (Q)SAR models. The modeling systems used were MultiCASE, Leadscope and MDL QSAR and the developed models cross...

  20. The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

    Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V; Bastien, Dominic; Bérubé, Gervais; Sligar, Stephen G; Kincaid, James R

    2016-05-01

    Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. PMID:26774838

  1. Molecular Docking of 3-Methylindole-containing Drugs Binding into CYP3A4

    MENG Xuan-yu; LI Zhuo; NIU Rui-juan; ZHANG Hong-xing; ZHENG Qing-chuan

    2012-01-01

    Drugs SPD-304(6,7-dimethyl-3- { [methyl-(2-{methyl-[ 1-(3-trifluoromethyl-phenyl)- 1H-indol-3-ylmethyl]-amino}-ethyl)-amino]-methyl}-chromen-4-one) and zafirlukast contain a common structural element of 3-substituted indole moiety which closely relates to a dehydrogenated reaction catalyzed by cytochrome P450s(CYPs).It was reported that the dehydrogenation can produce a reactive electrophilic intermediate which cause toxicities and inactivate CYPs. Drug L-745,870(3-{[4-(4-chlorophenyl)piperazin-l-yl]-methyl}-1H-pyrrolo-2,3-β-pyridine) might have similar effect since it contains the same structural element.We used molecular docking approach combined with molecular dynamics(MD) simulation to model three-dimensional(3D) complex structures of SPD-304,zafirlukast and L-745,870 into CYP3A4,respectively.The results show that these three drugs can stably bind into the active site and the 3-methylene carbons of the drugs keep a reasonable reactive distance from the heme iron.The complex structure of SPD-304-CYP3A4 is in agreement with experimental data.For zafirlukast,the calculation results indicate that 3-methylene carbon might be the dehydrogenation reaction site.Docking model of L-745,870-CYP3A4 shows a potential possibility of L-745,870 dehydrogenated by CYP3A4 at 3-methylene carbon which is in agreement with experiment in vivo.In addition,residues in the phenylalanine cluster as well as S119 and R212 play a critical role in the ligands binding based on our calculations.The docking models could provide some clues to understand the metabolic mechanism of the drugs by CYP3A4.

  2. Potential implications of CYP3A4, CYP3A5 and MDR-1 genetic variants on the efficacy of Lopinavir/Ritonavir (LPV/r monotherapy in HIV-1 patients

    Giulia Berno

    2014-11-01

    Full Text Available Introduction: Several genetic single nucleotide polymorphisms (SNPs in biotransformation enzymes (CYP3A4, CYP3A5 or transporter proteins (multidrug resistance MDR1 gene product, P-gp are involved in PI metabolism so that PI pharmacokinetics is characterized by a large inter-individual variability. The aim of this study was: (i to develop an in-house PCR/direct sequencing, based on DNA purification of full-length CYP3A4 and CYP3A5 genes (SNPs and MDR1 C3435T variant; (ii to investigate association of CYP3A4 and CYP3A5 reported or unreported genetic polymorphisms and MDR1-C3435T (CC homozygote, CT heterozygote, TT homozygote with clinical outcome of HIV-1 infected subjects treated with PI. Methods: Overall, 39 HIV-1 infected patients receiving boosted Lopinavir (LPV/r monotherapy after virological suppression were genotyped and analyzed through PCR and direct sequencing of full-length CYP3A4 and CYP3A5 gene sequences (1 and MDR1 gene (C3435T. CD4+T-cell counts and plasma viral load were analyzed before and after LPV/r initiation; LPV/r therapeutic drug monitoring (TDM was determined at 12-hours. Results: LPV/r TDM (ng/ml did not show significant differences among CYP3A4 or CYP3A5 SNPs, although a mean lower level of LPV/r was associated with detection of several SNPs: CYP3A5*3 rs776746; CYP3A5 rs28365088, CYP3A5 rs15524, CYP3A4 rs2687116, and a not already described polymorphism CYP3A4 nt20338. In follow-up analysis, <90% adherence was the main factor associated with virological failure of LPV/r monotherapy (83.3% of failure vs 34.4%, p<0.001 at log-rank test. Adjusting for adherence, the detection of a single CYP3A5*3 rs776746 and CYP3A5 rs15524 SNPs was associated with higher probability of LPV/r monotherapy failure (p<0.01, and in general, detection of any CYP3A5 SNP was associated with failure (26.2% vs 58.3%, p=0.067. No-association with detection of any CYP3A4 SNPs was found. MDR1 TT variants showed significant lower frequency of treatment

  3. Modeling Chemical Interaction Profiles: II. Molecular Docking, Spectral Data-Activity Relationship, and Structure-Activity Relationship Models for Potent and Weak Inhibitors of Cytochrome P450 CYP3A4 Isozyme

    Eugene Demchuk

    2012-03-01

    Full Text Available Polypharmacy increasingly has become a topic of public health concern, particularly as the U.S. population ages. Drug labels often contain insufficient information to enable the clinician to safely use multiple drugs. Because many of the drugs are bio-transformed by cytochrome P450 (CYP enzymes, inhibition of CYP activity has long been associated with potentially adverse health effects. In an attempt to reduce the uncertainty pertaining to CYP-mediated drug-drug/chemical interactions, an interagency collaborative group developed a consensus approach to prioritizing information concerning CYP inhibition. The consensus involved computational molecular docking, spectral data-activity relationship (SDAR, and structure-activity relationship (SAR models that addressed the clinical potency of CYP inhibition. The models were built upon chemicals that were categorized as either potent or weak inhibitors of the CYP3A4 isozyme. The categorization was carried out using information from clinical trials because currently available in vitro high-throughput screening data were not fully representative of the in vivo potency of inhibition. During categorization it was found that compounds, which break the Lipinski rule of five by molecular weight, were about twice more likely to be inhibitors of CYP3A4 compared to those, which obey the rule. Similarly, among inhibitors that break the rule, potent inhibitors were 2–3 times more frequent. The molecular docking classification relied on logistic regression, by which the docking scores from different docking algorithms, CYP3A4 three-dimensional structures, and binding sites on them were combined in a unified probabilistic model. The SDAR models employed a multiple linear regression approach applied to binned 1D 13C-NMR and 1D 15N-NMR spectral descriptors. Structure-based and physical-chemical descriptors were used as the basis for developing SAR models by the decision forest method. Thirty-three potent inhibitors

  4. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  5. Vitamin K2 in Electron Transport System: Are Enzymes Involved in Vitamin K2 Biosynthesis Promising Drug Targets?

    Eeshwaraiah Begari

    2010-03-01

    Full Text Available Aerobic and anaerobic respiratory systemsallow cells to transport the electrons to terminal electron acceptors. The quinone (ubiquinone or menaquinone pool is central to the electron transport chain. In the majority of Gram-positive bacteria, vitamin K2 (menaquinone is the sole quinone in the electron transport chain, and thus, the bacterial enzymes catalyzing the synthesis of menaquinone are potential targets for the development of novel antibacterial drugs. This manuscript reviews the role of vitamin K in bacteria and humans, and especially emphasizes on recent aspects of menaquinones in bacterial electron transport chain and on discoveries of inhibitor molecules targeting bacterial electron transport systems for new antibacterial agents.

  6. Influence of fermentation conditions on polysaccharide production and the activities of enzymes involved in the polysaccharide synthesis of Cordyceps militaris.

    Zhu, Zhen-Yuan; Liu, Xiao-Cui; Dong, Feng-Ying; Guo, Ming-Zhu; Wang, Xiao-Ting; Wang, Zheng; Zhang, Yong-Min

    2016-05-01

    The influence of different fermentation conditions on intracellular polysaccharide (IPS) production and activities of the phosphoglucomutase (PGM), UDPG-pyrophosphorylase (UGP), phosphoglucose isomerase (PGI), UDPG-dehydrogenase (UGD), and glucokinase (GK) implicated in metabolite synthesis in Cordyceps militaris was evaluated. The highest IPS production (327.57 ± 6.27 mg/100 mL) was obtained when the strain was grown in the optimal medium containing glucose (40 g · L(-1)), beef extract (10 g · L(-1)), and CaCO3 (0.5 g · L(-1)), and the initial pH and temperature were 7 and 25 °C, respectively. The activities of PGM, UGP, and PGI were proved to be influenced by the fermentation conditions. A strong correlation between the activities of these enzymes and the production of IPS was found. The transcription level of the pgm gene (encoding PGM) was 1.049 times and 1.467 times compared to the ugp gene and pgi gene (encoding UGP and PGI), respectively, in the optimal culture medium. This result indicated that PGM might be the highly key enzyme to regulate the biosynthesis of IPS of C. militaris in a liquid-submerged culture. Our study might be helpful for further research on the pathway of polysaccharide biosynthesis aimed to improve the IPS production of C. militaris. PMID:26685672

  7. Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins

    Amy V. Callaghan

    2013-05-01

    Full Text Available Anaerobic microorganisms play key roles in the biogeochemical cycling of methane and non-methane alkanes. To date, there appear to be at least three proposed mechanisms of anaerobic methane oxidation (AOM. The first pathway is mediated by consortia of archaeal anaerobic methane oxidizers and sulfate-reducing bacteria via ‘reverse methanogenesis’ and is catalyzed by a homologue of methyl-coenzyme M reductase. The second pathway is also mediated by anaerobic methane oxidizers and sulfate-reducing bacteria, wherein the archaeal members catalyze both methane oxidation and sulfate reduction and zero-valent sulfur is a key intermediate. The third AOM mechanism is a nitrite-dependent, intra-aerobic pathway described for the denitrifying bacterium, ‘Candidatus Methylomirabilis oxyfera.’ It is hypothesized that AOM proceeds via reduction of nitrite to nitric oxide, followed by the conversion of two nitric oxide molecules to dinitrogen and molecular oxygen. The latter can be used to functionalize the methane via a particulate methane monooxygenase. With respect to non-methane alkanes, there also appears to be novel mechanisms of activation. The most well-described pathway is the addition of non-methane alkanes across the double bond of fumarate to form alkyl-substituted succinates via the putative glycyl radical enzyme, alkylsuccinate synthase (also known as methylalkylsuccinate synthase. Other proposed mechanisms include anaerobic hydroxylation via ethylbenzene dehydrogenase-like enzymes and an ‘intra-aerobic’ denitrification pathway similar to that described for ‘M. oxyfera.’

  8. Polyphenols rich fraction from Geoffroea decorticans fruits flour affects key enzymes involved in metabolic syndrome, oxidative stress and inflammatory process.

    Costamagna, M S; Zampini, I C; Alberto, M R; Cuello, S; Torres, S; Pérez, J; Quispe, C; Schmeda-Hirschmann, G; Isla, M I

    2016-01-01

    Geoffroea decorticans (chañar), is widely distributed throughout Northwestern Argentina. Its fruit is consumed as flour, arrope or hydroalcoholic beverage. The chañar fruits flour was obtained and 39 phenolic compounds were tentatively identified by HPLC-MS/MS(n). The compounds comprised caffeic acid glycosides, simple phenolics (protocatechuic acid and vanillic acid), a glycoside of vanillic acid, p-coumaric acid and its phenethyl ester as well as free and glycosylated flavonoids. The polyphenols enriched extract with and without gastroduodenal digestion inhibited enzymes associated with metabolic syndrome, including α-amylase, α-glucosidase, lipase and hydroxyl methyl glutaryl CoA reductase. The polyphenolic extract exhibited antioxidant activity by different mechanisms and inhibited the pro-inflammatory enzymes (ciclooxygenase, lipoxygenase and phospholipase A2). The polyphenolic extract did not showed mutagenic effect by Ames test against Salmonella typhimurium TA98 and TA100 strains. These findings add evidence that chañar fruit flour may be considered a functional food with preventive properties against diseases associated with oxidative stress, inflammatory mediators and metabolic syndrome. PMID:26212988

  9. Human cytochrome P450 3A4 and a carbon nanofiber modified film electrode as a platform for the simple evaluation of drug metabolism and inhibition reactions.

    Xue, Qiang; Kato, Dai; Kamata, Tomoyuki; Guo, Qiaohui; You, Tianyan; Niwa, Osamu

    2013-11-01

    Electrochemical biosensors consisting of cytochrome P450 enzyme modified electrodes have been developed to provide a simple method for screening the metabolism of a drug and its inhibitor. Here, we report a very simple electrochemically driven biosensor for detecting drug metabolism and its inhibition based on cytochrome P450 3A4 (CYP3A4) and a carbon nanofiber (CNF) modified film electrode without any other modified layers such as mediator films. Direct electron transfer (DET) between CYP3A4 and CNFs was observed at a formal potential of -0.302 V. The electrocatalytic reduction current increased with the addition of drugs including testosterone and quinidine. In contrast, the reduction current was greatly suppressed in the presence of ketoconazole, which is a CYP3A4 inhibitor. CNFs with high conductivity, a large surface area and sufficient edge planes provide a suitable microenvironment for achieving excellent DET and biocatalysis properties, which could not be observed when we used other carbon materials such as carbon nanotube (CNT) and carbon black (CB) modified electrodes, indicating that our system is promising as a new bioelectronic platform for electrochemical biosensing. PMID:24027778

  10. An efficient method for following the enzymic reactions involved in camphor biosynthesis in Cinnamomum camphora by use of GC-MS and regiospecifically deuteriated substrate

    Yamamitsu, Tohru; Suga, Takayuki (Hiroshima Univ., Kagamiyama (Japan). Dept. of Chemistry); Ohta, Shinji (Hiroshima Univ., Higashisenda-machi, Naka-ku (Japan). Instrument Center for Chemical Analysis)

    1992-05-01

    An efficient method has been developed to follow the enzymic reactions involved in the biosynthesis of camphor in Cinnamomum camphora (camphor tree) by use of (5,5-{sup 2}H{sub 2})geranyl diphosphate as a substrate and GC-MS with selected ion monitoring. Borneol and camphor biosynthesized in the enzymic reactions gave a base peak due to the deuterium-containing ion in its EI mass spectrum. It is possible to detect 1.5 ng of the biosynthesized borneol and camphor per injection into GC-MS. This method enabled us to differentiate easily the biosynthesized camphor from the endogenous camphor and it is a facile and sensitive technique to determine the amount of the biosynthesized camphor. (author).

  11. An efficient method for following the enzymic reactions involved in camphor biosynthesis in Cinnamomum camphora by use of GC-MS and regiospecifically deuteriated substrate

    An efficient method has been developed to follow the enzymic reactions involved in the biosynthesis of camphor in Cinnamomum camphora (camphor tree) by use of [5,5-2H2]geranyl diphosphate as a substrate and GC-MS with selected ion monitoring. Borneol and camphor biosynthesized in the enzymic reactions gave a base peak due to the deuterium-containing ion in its EI mass spectrum. It is possible to detect 1.5 ng of the biosynthesized borneol and camphor per injection into GC-MS. This method enabled us to differentiate easily the biosynthesized camphor from the endogenous camphor and it is a facile and sensitive technique to determine the amount of the biosynthesized camphor. (author)

  12. Receptor-dependent regulation of the CYP3A4 gene

    A CYP3A4 promoter-reporter gene construct has been used to assess the ability of 16 known (in vivo) and putative (in vitro) inducers to transactivate a CYP3A4 reporter gene in HepG2 cells. With the exception of pravastatin, the remaining 15 compounds transactivated the CYP3A4 reporter gene with differing inductive abilities (Imax:EC50) over two orders of magnitude, ranging from 1.1 (phenytoin) to 222.9 (lovastatin) in a receptor-supplemented system and it is proposed that the lack of response to pravastatin is due to loss of the known hepatic uptake transporter in HepG2 cells. In addition, reporter gene assays were used to investigate two promoter mutants namely a T to C change at -191 bp in the hepatic nuclear factor 3 binding site (HNF-3, -187 to -194 bp) and an A to G change at -205 bp in the oestrogen response element (ERE, -202 to -212 bp), which conferred differential responsiveness to steroid and xenobiotic inducers

  13. Effects of the CYP3A4*1B Genetic Polymorphism on the Pharmacokinetics of Tacrolimus in Adult Renal Transplant Recipients: A Meta-Analysis.

    Wei-Long Shi

    Full Text Available The association between the CYP3A4*1B single nucleotide polymorphism (SNP and tacrolimus pharmacokinetics in different studies is controversial. Therefore, a meta-analysis was employed to evaluate the correlation between the CYP3A4*1B genetic polymorphism and tacrolimus pharmacokinetics at different post-transplantation times in adult renal transplant recipients.Studies evaluating the CYP3A4*1B genetic polymorphism and tacrolimus pharmacokinetics were retrieved through a systematical search of Embase, PubMed, the Cochrane Library, ClinicalTrials.gov and three Chinese literature databases (up to Sept. 2014. The pharmacokinetic parameters (weight-adjusted tacrolimus daily dose and tacrolimus trough concentration/weight-adjusted tacrolimus daily dose ratio were extracted, and the meta-analysis was performed using Stata 12.1.Seven studies (involving 1182 adult renal transplant recipients were included in this meta-analysis. For the weight-adjusted tacrolimus daily dose, in all included renal transplant recipients (European & Indian populations, CYP3A4*1/*1 recipients required a significantly lower weight-adjusted tacrolimus daily dose than did CYP3A4*1B carriers at 7 days (WMD -0.048; 95% CI -0.083 ~ -0.014, 6 months (WMD -0.058; 95% CI -0.081 ~ -0.036 and 12 months (WMD - 0.061; 95% CI -0.096 ~ -0.027 post-transplantation. In light of the heterogeneity, the analysis was repeated after removing the only study in an Indian population, and CYP3A4*1/*1 European recipients (mostly Caucasian required a lower weight-adjusted tacrolimus daily dose within the first year post-transplantation. The tacrolimus trough concentration/weight-adjusted tacrolimus daily dose ratio (C0/Dose ratio was significantly higher in CYP3A4*1/*1 recipients than in CYP3A4*1B carriers at 6 months (WMD 52.588; 95% CI 22.387 ~ 82.789 and 12 months (WMD 62.219; 95% CI 14.218 ~ 110.221 post-transplantation. When the only study in an Indian population was removed to examine European

  14. Bone marrow involvement in Gaucher disease at MRI: what long-term evolution can we expect under enzyme replacement therapy?

    Fedida, Benjamin; Touraine, Sebastien; Laredo, Jean-Denis [Hopital Lariboisiere, AP-HP, Department of Musculoskeletal Imaging, Paris (France); Stirnemann, Jerome [Universite Paris-Diderot Hopital Bichat, AP-HP, Department of Biostatistics and Medical Data Processing, INSERM UMR 738, Paris (France); Geneva University Hospital, Division of General Internal Medicine, Faculty of Medicine, Geneva (Switzerland); Belmatoug, Nadia [Hopital Beaujon, AP-HP, Referral Center for Lysosomal Diseases (RCLD), Clichy (France); Hopital Beaujon, AP-HP, Department of Internal Medicine, Clichy (France); Petrover, David [Hopital Lariboisiere, AP-HP, Department of Musculoskeletal Imaging, Paris (France); Hopital Beaujon, AP-HP, Referral Center for Lysosomal Diseases (RCLD), Clichy (France)

    2015-10-15

    To study the long-term evolution of the bone marrow burden (BMB) score at MRI in patients with Gaucher disease (GD) under enzyme replacement therapy (ERT). Forty patients treated for GD were retrospectively studied in a referral centre. BMB scores were assessed on spine and femur MR examinations performed between January 2003 and June 2014. The long-term evolution of the BMB scores was analyzed using a linear mixed model. A total of 121 MRI examinations were performed during the study period with a mean follow-up of 7.1 years ± 5.6, an average rate of 3.1 MR examinations ± 1.7 per patient and an interval of 2.3 years ± 1.1 between examinations. Patients had received ERT during 12 years on average ± 6.7. The trend of BMB scores with time decreased significantly by 15 % (P = 0.008) during the total study period and 39 % (P = 0.01) during the first 5 years of treatment. No changes in BMB scores were observed after five years of treatment. In Gaucher patients, the trend of MRI BMB scores with time decreased significantly under ERT the first 5 years of treatment before a long-term stabilization. (orig.)

  15. Possible Involvement of Reactive Oxygen Species Scavenging Enzymes in Desiccation Sensitivity of Antiaris toxicaria Seeds and Axes

    Hong-Yan Cheng; Song-Quan Song

    2008-01-01

    The relationships among desiccation sensitivities of Antiaris toxicaria seeds and axes, changes in activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase, (TBA)-reactive substance were studied. Desiccation tolerance of seeds and axes decreased with dehydration. Desiccation tolerance of axes was higher than that of seeds, and that of epicotyls was higher than radicles. Activities of SOD, CAT and DHAR of seeds increased during the initial phase of dehydration, and then decreased with further dehydration, whereas activities of APX and GR decreased with dehydration. These five enzyme activities of axes, however, increased during the initial phase of dehydration, and then decreased with further dehydration. The rate of superoxide radical production, and the contents of H2O2 and TBA-reactive products of seeds and axes gradually increased with dehydration. These results show that the A. toxicaria seed is a typical recalcitrant seed. Loss of desiccation tolerance in seeds and axes was correlated with activities of seeds and axes.

  16. Beyond glycolysis: GAPDHs are multi-functional enzymes involved in regulation of ROS, autophagy, and plant immune responses.

    Elizabeth Henry

    2015-04-01

    Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is an important enzyme in energy metabolism with diverse cellular regulatory roles in vertebrates, but few reports have investigated the importance of plant GAPDH isoforms outside of their role in glycolysis. While animals possess one GAPDH isoform, plants possess multiple isoforms. In this study, cell biological and genetic approaches were used to investigate the role of GAPDHs during plant immune responses. Individual Arabidopsis GAPDH knockouts (KO lines exhibited enhanced disease resistance phenotypes upon inoculation with the bacterial plant pathogen Pseudomonas syringae pv. tomato. KO lines exhibited accelerated programmed cell death and increased electrolyte leakage in response to effector triggered immunity. Furthermore, KO lines displayed increased basal ROS accumulation as visualized using the fluorescent probe H2DCFDA. The gapa1-2 and gapc1 KOs exhibited constitutive autophagy phenotypes in the absence of nutrient starvation. Due to the high sequence conservation between vertebrate and plant cytosolic GAPDH, our experiments focused on cytosolic GAPC1 cellular dynamics using a complemented GAPC1-GFP line. Confocal imaging coupled with an endocytic membrane marker (FM4-64 and endosomal trafficking inhibitors (BFA, Wortmannin demonstrated cytosolic GAPC1 is localized to the plasma membrane and the endomembrane system, in addition to the cytosol and nucleus. After perception of bacterial flagellin, GAPC1 dynamically responded with a significant increase in size of fluorescent puncta and enhanced nuclear accumulation. Taken together, these results indicate that plant GAPDHs can affect multiple aspects of plant immunity in diverse sub-cellular compartments.

  17. Beyond glycolysis: GAPDHs are multi-functional enzymes involved in regulation of ROS, autophagy, and plant immune responses.

    Henry, Elizabeth; Fung, Nicholas; Liu, Jun; Drakakaki, Georgia; Coaker, Gitta

    2015-04-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme in energy metabolism with diverse cellular regulatory roles in vertebrates, but few reports have investigated the importance of plant GAPDH isoforms outside of their role in glycolysis. While animals possess one GAPDH isoform, plants possess multiple isoforms. In this study, cell biological and genetic approaches were used to investigate the role of GAPDHs during plant immune responses. Individual Arabidopsis GAPDH knockouts (KO lines) exhibited enhanced disease resistance phenotypes upon inoculation with the bacterial plant pathogen Pseudomonas syringae pv. tomato. KO lines exhibited accelerated programmed cell death and increased electrolyte leakage in response to effector triggered immunity. Furthermore, KO lines displayed increased basal ROS accumulation as visualized using the fluorescent probe H2DCFDA. The gapa1-2 and gapc1 KOs exhibited constitutive autophagy phenotypes in the absence of nutrient starvation. Due to the high sequence conservation between vertebrate and plant cytosolic GAPDH, our experiments focused on cytosolic GAPC1 cellular dynamics using a complemented GAPC1-GFP line. Confocal imaging coupled with an endocytic membrane marker (FM4-64) and endosomal trafficking inhibitors (BFA, Wortmannin) demonstrated cytosolic GAPC1 is localized to the plasma membrane and the endomembrane system, in addition to the cytosol and nucleus. After perception of bacterial flagellin, GAPC1 dynamically responded with a significant increase in size of fluorescent puncta and enhanced nuclear accumulation. Taken together, these results indicate that plant GAPDHs can affect multiple aspects of plant immunity in diverse sub-cellular compartments. PMID:25918875

  18. Bone marrow involvement in Gaucher disease at MRI: what long-term evolution can we expect under enzyme replacement therapy?

    To study the long-term evolution of the bone marrow burden (BMB) score at MRI in patients with Gaucher disease (GD) under enzyme replacement therapy (ERT). Forty patients treated for GD were retrospectively studied in a referral centre. BMB scores were assessed on spine and femur MR examinations performed between January 2003 and June 2014. The long-term evolution of the BMB scores was analyzed using a linear mixed model. A total of 121 MRI examinations were performed during the study period with a mean follow-up of 7.1 years ± 5.6, an average rate of 3.1 MR examinations ± 1.7 per patient and an interval of 2.3 years ± 1.1 between examinations. Patients had received ERT during 12 years on average ± 6.7. The trend of BMB scores with time decreased significantly by 15 % (P = 0.008) during the total study period and 39 % (P = 0.01) during the first 5 years of treatment. No changes in BMB scores were observed after five years of treatment. In Gaucher patients, the trend of MRI BMB scores with time decreased significantly under ERT the first 5 years of treatment before a long-term stabilization. (orig.)

  19. Fatty acid hydroperoxide lyase : a plant cytochrome P450 enzyme involved in wound healing and pest resistance

    Vliegenthart, J. F. G.; Noordermeer, M.A.; Veldink, G.A.

    2001-01-01

    Plants continuously have to defend themselves against life-threatening events such as drought, mechanical damage, temperature stress, and potential pathogens. Nowadays, more and more similarities between the defense mechanism of plants and that of animals are being discovered. In both cases, the lipoxygenase pathway plays an important role. In plants, products of this pathway are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity. The...

  20. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment

  1. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States); Schuetz, Erin G. [Department of Pharmaceutical Sciences, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States); Chen, Taosheng, E-mail: taosheng.chen@stjude.org [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States)

    2013-10-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment.

  2. Right ventricular involvement in patients with Fabry's disease and the effect of enzyme replacement therapy

    Wuest, W. [Universitaetsklinikum Erlangen (Germany). Radiologisches Inst.; Machann, W.; Koestler, H.; Hahn, D.; Beer, M. [Universitaetsklinikum Wuerzburg (Germany). Inst. fuer Roentgendiagnostik; Breunig, F.; Weidemann, F.; Wanner, C. [Universitaetsklinikum Wuerzburg (Germany). Medizinische Klinik I

    2011-11-15

    According to echocardiography reports, Fabry cardiomyopathy not only affects the left ventricle (LV) but also the right ventricle (RV). Until now no MRI studies about the effect of enzyme replacement therapy (ERT) on the RV are available. We evaluated the effect of ERT on the RV. In this prospective trial 14 patients with genetically proven Fabry's disease were examined using a 1.5 T MR scanner before ERT and after 13 {+-} 1 months of ERT. All patients underwent cardiac MR imaging and the RV/LV cardiac morphology and function were analyzed. At baseline examination the values were as follows: RV mass 31 {+-} 6 g/m{sup 2}, end-diastolic volume (EDV) 88 {+-} 13 ml/m{sup 2}, end-systolic volume (ESV) 39 {+-} 9 ml/m{sup 2}, stroke volume (SV) 49 {+-} 7 ml/m{sup 2} and ejection fraction (EF) 56 {+-} 5 %. The RV mass and EDV decreased significantly after 13 {+-} 1 months on ERT (mass 27 {+-} 7 g/m{sup 2}, p < 0.05, EDV 76 {+-} 24 ml/m{sup 2}, p < 0.05), with no significant change of ESV (33 {+-} 13 ml/m{sup 2}), SV (43 {+-} 12 ml/m{sup 2}) and EF (57 {+-} 7 %). The LV mass (102 {+-} 26 g/m{sup 2} vs. 94 {+-} 27 g/m{sup 2}, p < 0.05), EDV (76 {+-} 13 ml/m{sup 2} vs. 66 {+-} 22 ml/m{sup 2}, p < 0.05) and ESV (29 {+-} 9 ml/m{sup 2} vs. 23 {+-} 9 ml/m{sup 2}, p < 0.05) decreased significantly while the EF (64 {+-} 7 % vs. 66 {+-} 5 %; p < 0.05) increased significantly. Besides the known beneficial effect on the LV, ERT improves RV mass and EDV. (orig.)

  3. Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

    Siyun Xu

    2014-10-01

    Full Text Available RNA interference (RNAi is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4, which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3 were designed to target the coding sequence (CDS of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55% in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.

  4. Two herbivore-induced cytochrome P450 enzymes CYP79D6 and CYP79D7 catalyze the formation of volatile aldoximes involved in poplar defense.

    Irmisch, Sandra; McCormick, Andrea Clavijo; Boeckler, G Andreas; Schmidt, Axel; Reichelt, Michael; Schneider, Bernd; Block, Katja; Schnitzler, Jörg-Peter; Gershenzon, Jonathan; Unsicker, Sybille B; Köllner, Tobias G

    2013-11-01

    Aldoximes are known as floral and vegetative plant volatiles but also as biosynthetic intermediates for other plant defense compounds. While the cytochrome P450 monooxygenases (CYP) from the CYP79 family forming aldoximes as biosynthetic intermediates have been intensively studied, little is known about the enzymology of volatile aldoxime formation. We characterized two P450 enzymes, CYP79D6v3 and CYP79D7v2, which are involved in herbivore-induced aldoxime formation in western balsam poplar (Populus trichocarpa). Heterologous expression in Saccharomyces cerevisiae revealed that both enzymes produce a mixture of different aldoximes. Knockdown lines of CYP79D6/7 in gray poplar (Populus × canescens) exhibited a decreased emission of aldoximes, nitriles, and alcohols, emphasizing that the CYP79s catalyze the first step in the formation of a complex volatile blend. Aldoxime emission was found to be restricted to herbivore-damaged leaves and is closely correlated with CYP79D6 and CYP79D7 gene expression. The semi-volatile phenylacetaldoxime decreased survival and weight gain of gypsy moth (Lymantria dispar) caterpillars, suggesting that aldoximes may be involved in direct defense. The wide distribution of volatile aldoximes throughout the plant kingdom and the presence of CYP79 genes in all sequenced genomes of angiosperms suggest that volatile formation mediated by CYP79s is a general phenomenon in the plant kingdom. PMID:24220631

  5. Analysis of CYP3A4*1B and CYP3A5*3 polymorphisms in population of Bosnia and Herzegovina

    Sabina Semiz

    2011-02-01

    Full Text Available Aim Differences in the frequency of distribution of the cytochromeP450 (CYP allelic variants have been demonstrated between distinct ethnic groups, contributing to observed interindividual variation in drug response. In this study we determined, for the irst time, prevalence of the common allelic variants of the polymorphic CYP enzymes, CYP3A4*1B and CYP3A5*3, in the population of Bosnia and Herzegovina (BH. Methods Genomic DNA was extracted from blood samples collected from 140 unrelated subjects. A real-time PCR was used for the detection of CYP polymorphisms, with the application of the speciic TaqMan® SNP Genotyping Assay (Applied Biosystemsfor CYP3A5*3, while CYP3A4*1B was genotyped by high-resolution melting analysis. Results Our results have shown that the distribution of CYP3A4*1B and CYP3A5*3 alleles was in line with the data reported in European Caucasians. We conirmed that CYP3A4*1B mutant allele is rare in Caucasians, being present in only 5.1% individuals. However, CYP3A5*3 polymorphism was found to be predominant in the Bosnian population with an incidence of 94%, similarly to other European populations tested so far. Interestingly, we have demonstrated a strong linkage disequilibrium between CYP3A5*3 and CYP3A4*1B alleles. No signiicant difference in allele frequencies for CYP3A4*1B and CYP3A5*3 has been shown between male and female subjects participating in our study. Conclusion Our data demonstrated the high prevalence of CYP3A5*3 allele in Bosnian population, indicating signiicance of analysis of CYP3A5 and CYP3A4 polymorphisms and corresponding allele frequencies in speciic ethnic groups. Importantly, results of this study may lead to translation of pharmacogenetics and individualized therapeutic approach in current clinical practices in BH.

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

    Richard D. Beger

    2012-03-01

    Full Text Available An interagency collaboration was established to model chemical interactions that may cause adverse health effects when an exposure to a mixture of chemicals occurs. Many of these chemicals—drugs, pesticides, and environmental pollutants—interact at the level of metabolic biotransformations mediated by cytochrome P450 (CYP enzymes. In the present work, spectral data-activity relationship (SDAR and structure-activity relationship (SAR approaches were used to develop machine-learning classifiers of inhibitors and non-inhibitors of the CYP3A4 and CYP2D6 isozymes. The models were built upon 602 reference pharmaceutical compounds whose interactions have been deduced from clinical data, and 100 additional chemicals that were used to evaluate model performance in an external validation (EV test. SDAR is an innovative modeling approach that relies on discriminant analysis applied to binned nuclear magnetic resonance (NMR spectral descriptors. In the present work, both 1D 13C and 1D 15N-NMR spectra were used together in a novel implementation of the SDAR technique. It was found that increasing the binning size of 1D 13C-NMR and 15N-NMR spectra caused an increase in the tenfold cross-validation (CV performance in terms of both the rate of correct classification and sensitivity. The results of SDAR modeling were verified using SAR. For SAR modeling, a decision forest approach involving from 6 to 17 Mold2 descriptors in a tree was used. Average rates of correct classification of SDAR and SAR models in a hundred CV tests were 60% and 61% for CYP3A4, and 62% and 70% for CYP2D6, respectively. The rates of correct classification of SDAR and SAR models in the EV test were 73% and 86% for CYP3A4, and 76% and 90% for CYP2D6, respectively. Thus, both SDAR and SAR methods demonstrated a comparable performance in modeling a large set of structurally diverse data. Based on unique NMR structural descriptors, the new SDAR modeling method complements the existing SAR

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

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

    2012-01-01

    An interagency collaboration was established to model chemical interactions that may cause adverse health effects when an exposure to a mixture of chemicals occurs. Many of these chemicals--drugs, pesticides, and environmental pollutants--interact at the level of metabolic biotransformations mediated by cytochrome P450 (CYP) enzymes. In the present work, spectral data-activity relationship (SDAR) and structure-activity relationship (SAR) approaches were used to develop machine-learning classifiers of inhibitors and non-inhibitors of the CYP3A4 and CYP2D6 isozymes. The models were built upon 602 reference pharmaceutical compounds whose interactions have been deduced from clinical data, and 100 additional chemicals that were used to evaluate model performance in an external validation (EV) test. SDAR is an innovative modeling approach that relies on discriminant analysis applied to binned nuclear magnetic resonance (NMR) spectral descriptors. In the present work, both 1D ¹³C and 1D ¹⁵N-NMR spectra were used together in a novel implementation of the SDAR technique. It was found that increasing the binning size of 1D ¹³C-NMR and ¹⁵N-NMR spectra caused an increase in the tenfold cross-validation (CV) performance in terms of both the rate of correct classification and sensitivity. The results of SDAR modeling were verified using SAR. For SAR modeling, a decision forest approach involving from 6 to 17 Mold2 descriptors in a tree was used. Average rates of correct classification of SDAR and SAR models in a hundred CV tests were 60% and 61% for CYP3A4, and 62% and 70% for CYP2D6, respectively. The rates of correct classification of SDAR and SAR models in the EV test were 73% and 86% for CYP3A4, and 76% and 90% for CYP2D6, respectively. Thus, both SDAR and SAR methods demonstrated a comparable performance in modeling a large set of structurally diverse data. Based on unique NMR structural descriptors, the new SDAR modeling method complements the existing SAR

  8. Mutational analyses of the enzymes involved in the metabolism of hydrogen by the hyperthermophilic archaeon Pyrococcus furiosus

    Gerrit J Schut

    2012-05-01

    Full Text Available Pyrococcus furiosus grows optimally near 100°C by fermenting carbohydrates to produce hydrogen (H2 or, if elemental sulfur (S0, is present hydrogen sulfide instead. It contains two cytoplasmic hydrogenases, SHI and SHII, that use NADP(H as an electron carrier, and a membrane bound hydrogenase (MBH, that utilizes the redox protein ferredoxin. We previously constructed deletion strains lacking SHI and/or SHII and showed that they exhibited no obvious phenotype. This study has now been extended to include biochemical analyses and growth studies using the ΔSHI and ΔSHII deletion strains together with strains lacking a functional MBH (ΔMbhL. Hydrogenase activities in cytoplasmic extracts of ΔSHII and the parent strain were similar but were much lower (<10% in the ΔSHI strain, and no activity was detected in the ΔSHIΔSHII double deletion strain, indicating that SHI is responsible for most of the cytoplasmic hydrogenase activity. In contrast, the ΔmbhL strain showed no growth in the absence of S0, confirming the hypothesis that, in the absence of S0, MBH is the only enzyme that can dispose of reductant (as H2 generated during sugar oxidation. The deletion strain devoid of all three hydrogenases also grew only in the presence of S0 and did not produce any detectable H2. When grown in the presence of limiting S0, both H2S and H2 were produced by the parent and ΔSHI/ΔSHII strains. A significant amount of H2 was also produced by the ΔmbhL strain, showing that SHI can produce H2 from NADPH in vivo, although this does not enable significant growth of ΔmbhL in the absence of S0. We propose that the physiological function of SHI is to recycle H2 and provide a link between external H2 and the intracellular pool of NADPH needed for biosynthesis. This likely has a distinct energetic advantage in the environment, but it is clearly not required for growth of the organism under the usual laboratory conditions. The function of SHII, however, remains

  9. Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I-NBOMe and 25I-NBOH

    Nielsen, Line Marie; Holm, Niels Bjerre; Leth-Petersen, Sebastian;

    2016-01-01

    INBOH were identified as CYP3A4 and CYP2D6, respectively. The compound 25I-NBOH was also liable to direct glucuronidation, which may diminish the impact of CYP2D6 genetic polymorphism. Users of 25I-NBOMe may be subject to drug-drug interactions (DDI) if 25I-NBOMe is taken with a strong CYP3A4 inhibitor...

  10. Mechanism-Based Inhibition of Recombinant Human Cytochrome P450 3A4 by Tomato Juice Extract

    須永, 克佳; 大川, 健一; 中村, 健一; 大久保, 温子; 原田, 園子; 津田, 整

    2012-01-01

    This study investigates whether tomato juice can inhibit cytochrome P450 (CYP) 3A4-mediated drug metabolism. Three commercially available, additive-free tomato juices, along with homogenized fresh tomato, were analyzed for their ability to inhibit testosterone 6β-hydroxylation activity using human recombinant CYP3A4. Results were compared to that of grapefruit juice. Ethyl acetate extracts of the tomato juices moderately reduced residual activity of CYP3A4 testosterone 6β-hydroxylation activi...

  11. Magic-Angle Spinning Solid-State NMR Spectroscopy of Nanodisc– Embedded Human CYP3A4

    Kijac, Aleksandra; Li, Ying; Sligar, Stephen G.; Rienstra, Chad M.

    2007-01-01

    Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not b...

  12. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

    Farah El Najjar

    Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

  13. Manganese-induced regulations in growth, yield formation, quality characters, rice aroma and enzyme involved in 2-acetyl-1-pyrroline biosynthesis in fragrant rice.

    Li, Meijuan; Ashraf, Umair; Tian, Hua; Mo, Zhaowen; Pan, Shenggang; Anjum, Shakeel Ahmad; Duan, Meiyang; Tang, Xiangru

    2016-06-01

    Micro-nutrient application is essential for normal plant growth while a little is known about manganese (Mn)-induced regulations in morpho-physiological attributes, aroma formation and enzyme involved in 2-acetyl-1-pyrroline (2-AP) biosynthesis in aromatic rice. Present study aimed to examine the influence of four levels of Mn i.e., Mn1 (100 mg MnSO4 pot(-1)), Mn2 (150 mg MnSO4 pot(-1)), Mn3 (200 mg MnSO4 pot(-1)), and Mn4 (250 mg MnSO4 pot(-1)) on the growth, yield formation, quality characters, rice aroma and enzyme involved in 2-acetyl-1-pyrroline biosynthesis in two fragrant rice cultivars i.e., Meixiangzhan and Nongxiang 18. Pots without Mn application were served as control (Ck). Each pot contained 15 kg of soil. Effects on agronomic characters, quality attributes, 2-AP contents and enzymes involved in 2-AP biosynthesis have been studied in early and late season rice. Results depicted that Mn improved rice growth, yield and related characters, and some quality attributes significantly. It further up-regulated proline, pyrroline-5-carboxylic acid (P5C) (precursors of 2-AP), soluble proteins and activities of proline dehydrogenase (ProDH), Δ(1) pyrroline-5-carboxylic acid synthetase (P5CS) ornithine aminotransferase (OAT) that led to enhanced 2-AP production in rice grains. Moreover, higher Mn levels resulted in increased grain Mn contents in both rice cultivars. Along with growth and yield improvement, Mn application significantly improved rice aromatic contents. Overall, Nongxiang 18 accumulated more 2-AP contents than Meixiangzhan in both seasons under Mn application. This study further explored the importance of Mn in rice aroma formation and signifies that micro-nutrients can play significant roles in rice aroma synthesis; however, intensive studies at molecular levels are still needed to understand the exact mechanisms of Mn to improve rice aroma formation. PMID:26995311

  14. Magic-angle spinning solid-state NMR spectroscopy of nanodisc-embedded human CYP3A4.

    Kijac, Aleksandra Z; Li, Ying; Sligar, Stephen G; Rienstra, Chad M

    2007-12-01

    Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because X-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solid-state NMR (SSNMR) of CYP3A4 (Delta3-12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields approximately 2.5 mg of the enzymatically active, uniformly 13C,15N-enriched CYP3A4 from 1 L of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The 13C line widths measured from 13C-13C 2D chemical shift correlation spectra are approximately 0.5 ppm. The secondary structure distribution within several amino acid types determined from 13C chemical shifts is consistent with the ligand-free X-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality. PMID:17985934

  15. FROG INTESTINAL PERFUSION TO EVALUATE DRUG PERMEABILITY: APPLICATION TO P-gp AND CYP3A4 SUBSTRATES

    Neelima eYerasi

    2015-07-01

    Full Text Available AbstractTo evaluate the reliability of using in situ frog intestinal perfusion technique for permeability assessment of carrier transported drugs which are also substrates for CYP enzymes. Single Pass Intestinal Perfusion (SPIP studies were performed in frogs of the species Rana tigrina using established method for rats with some modifications after inducing anesthesia. Effective permeability coefficient (Peff of losartan and midazolam was calculated in the presence and absence of inhibitors using the parallel-tube model. Peff of losartan when perfused alone was found to be 0.427 ± 0.27×10-4cm/s and when it was co-perfused with inhibitors, significant change in Peff was observed. Peff of midazolam when perfused alone was found to be 2.03 ± 0.07 × 10-4cm/s and when it was co-perfused with inhibitors, no significant change in Peff was observed. Comparison of Peff calculated in frog with that of other available models and also humans suggested that the Peff values are comparable and reflected well with human intestinal permeability. It is possible to determine the Peff value for compounds which are dual substrates of P-gp and CYP3A4 using in situ frog intestinal perfusion technique. The calculated Peff values correlated well with reported Peff values of probe drugs. comparison of the Peff value of losartan obtained with that of reported human’s Peff and Caco 2 cell data, and comparison of the Peff value of midazolam with that of reported rat’s Peff, we could conclude that SPIP from model can be reliably used in preclinical studies for permeability estimation. This model may represent a valuable alternative to the low speed and high cost of conventional animal models (typically rodents for the assessment of intestinal permeability.

  16. Distinct roles of Ape1 protein, an enzyme involved in DNA repair, in high or low linear energy transfer ionizing radiation-induced cell killing.

    Wang, Hongyan; Wang, Xiang; Chen, Guangnan; Zhang, Xiangming; Tang, Xiaobing; Park, Dongkyoo; Cucinotta, Francis A; Yu, David S; Deng, Xingming; Dynan, William S; Doetsch, Paul W; Wang, Ya

    2014-10-31

    High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy. PMID:25210033

  17. An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics

    Many adverse drug reactions are caused by the cytochrome P450 (CYP) dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-mediated toxicity and to improve safety of drug candidates, we developed two in vitro cell-based assays by combining an activating system (human CYP3A4) with target cells (HepG2 cells): in the first method we incubated microsomes containing cDNA-expressed CYP3A4 together with HepG2 cells; in the second approach HepG2 cells were transiently transfected with CYP3A4. In both assay systems, CYP3A4 catalyzed metabolism was found to be comparable to the high levels reported in hepatocytes. Both assay systems were used to study ten CYP3A4 substrates known for their potential to form metabolites that exhibit higher toxicity than the parent compounds. Several endpoints of toxicity were evaluated, and the measurement of MTT reduction and intracellular ATP levels were selected to assess cell viability. Results demonstrated that both assay systems are capable to metabolize the test compounds leading to increased toxicity, compared to their respective control systems. The co-incubation with the CYP3A4 inhibitor ketoconazole confirmed that the formation of reactive metabolites was CYP3A4 dependent. To further validate the functionality of the two assay systems, they were also used as a 'detoxification system' using selected compounds that can be metabolized by CYP3A4 to metabolites less toxic than their parent compounds. These results show that both assay systems can be used to screen for metabolic activation, or de-activation, which may be useful as a rapid and relatively inexpensive in vitro assay for the prediction of CYP3A4 metabolism-mediated toxicity

  18. Frequency and prognostic significant of CYP3A4-A-290G polymorphism in acute myeloid leukemia

    Gamal T. Ali

    2014-11-01

    Full Text Available Cytochrome P450 3A4 (CYP3A4 is the most plentiful cytochrome P450 in adult human liver and small intestine and is responsible for detoxification of more than 50% of drugs in addition to the metabolic deactivation and metabolism of many carcinogens. Polymorphism of CYP3A4-A-290G considered the only allele that appears to stimulate CYP3A4 expression and has been associated with a number of clinical phenotypes, including prostate cancer, breast cancer, leukemia and the early onset of puberty. In this study, we analyzed the presence of CYP3A4-A-290G polymorphism in 77 newly diagnosed AML cases and 72 healthy control using PCR/RFLP aiming to show CYP3A4-A-290G polymorphism pattern in acute myeloid leukemia patients, and its role in disease severity and progression. A highly statistically significant difference was found between the control and AML groups as regards the heterozygous genotype (p-value = 0.002 and increases the risk of AML 11.4-fold. Also there was a highly significant difference between the control and AML patients regarding variant allele (G in AG and GG genotypes (p-value 0.001 and increases the risk of AML 19-fold. No statistically significant association found between the CYP3A4-A-290G polymorphism and different clinical or laboratory parameters as well as an initial response to treatment, overall survival and the disease free survival. We concluded that CYP3A4-A-290G polymorphism is a genotypic factor that increases the CYP3A4 enzymatic activity and increases the risk of AML by 18.9-fold.

  19. Rapid LC-MS Drug Metabolite Profiling Using Microsomal Enzyme Bioreactors in a Parallel Processing Format

    Bajrami, Besnik; Zhao, Linlin; Schenkman, John B.; Rusling, James F.

    2009-01-01

    Silica nanoparticle bioreactors featuring thin films of enzymes and polyions were utilized in a novel high-throughput 96-well plate format for drug metabolism profiling. The utility of the approach was illustrated by investigating the metabolism of the drugs diclofenac (DCF), troglitazone (TGZ) and raloxifene, for which we observed known metabolic oxidation and bioconjugation pathways and turnover rates. A broad range of enzymes was included by utilizing human liver (HLM), rat liver (RLM) and bicistronic human-cyt P450 3A4 (bicis.-3A4) microsomes as enzyme sources. This parallel approach significantly shortens sample preparation steps compared to an earlier manual processing with nanoparticle bioreactors, allowing a range of significant enzyme reactions to be processed simultaneously. Enzyme turnover rates using the microsomal bioreactors were 2-3 fold larger compared to using conventional microsomal dispersions, most likely because of better accessibility of the enzymes. Ketoconazole (KET) and quinidine (QIN), substrates specific to cyt P450 3A enzymes, were used to demonstrate applicability to establish potentially toxic drug-drug interactions involving enzyme inhibition and acceleration. PMID:19904994

  20. Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis.

    Meng, Qiang; Chen, Xin-Li; Wang, Chang-Yuan; Liu, Qi; Sun, Hui-Jun; Sun, Peng-Yuan; Huo, Xiao-Kui; Liu, Zhi-Hao; Yao, Ji-Hong; Liu, Ke-Xin

    2015-03-15

    Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp) and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes. PMID:25655198

  1. Human hepatic metabolism of the anti-osteoporosis drug eldecalcitol involves sterol C4-methyl oxidase

    Yasuda, Kaori; Iwanaga, Yuasa; Ogawa, Kazuaki; Mano, Hiroki; Ueno, Sera; Kimoto, Shutaro; Ohta, Miho; Kamakura, Masaki; Ikushiro, Shinichi; Sakaki, Toshiyuki

    2015-01-01

    The metabolism of eldecalcitol (ED-71), a 2β-hydroxypropoxylated analog of the active form of vitamin D3 was investigated by using in vitro systems. ED-71 was metabolized to 1α,2β,25-trihydroxyvitamin D3 (1α,2β,25(OH)3D3) in human small intestine and liver microsomes. To identify the enzymes involved in this metabolism, we examined NADPH-dependent metabolism by recombinant P450 isoforms belonging to the CYP1, 2, and 3 families, and revealed that CYP3A4 had the activity. However, the CYP3A4 -s...

  2. Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis

    Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp) and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes. - Highlights: • AB23A has at least three roles in protection against ANIT-induced liver injury. • AB23A decreases Ntcp, and increases Bsep, Mrp2 and Mdr2 expression. • AB23A represses Cyp7a1 and Cyp8b1 through inducing Shp and Fgf15 expression. • AB23A increases bile acid metabolism through inducing Sult2a1 expression. • FXR activation is involved

  3. Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis

    Meng, Qiang; Chen, Xin-li; Wang, Chang-yuan; Liu, Qi; Sun, Hui-jun; Sun, Peng-yuan; Huo, Xiao-kui; Liu, Zhi-hao; Yao, Ji-hong; Liu, Ke-xin, E-mail: kexinliu@dlmedu.edu.cn

    2015-03-15

    Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp) and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes. - Highlights: • AB23A has at least three roles in protection against ANIT-induced liver injury. • AB23A decreases Ntcp, and increases Bsep, Mrp2 and Mdr2 expression. • AB23A represses Cyp7a1 and Cyp8b1 through inducing Shp and Fgf15 expression. • AB23A increases bile acid metabolism through inducing Sult2a1 expression. • FXR activation is involved

  4. SPR and electrochemical analyses of interactions between CYP3A4 or 3A5 and cytochrome b5

    Gnedenko, O. V.; Yablokov, E. O.; Usanov, S. A.; Mukha, D. V.; Sergeev, G. V.; Bulko, T. V.; Kuzikov, A. V.; Moskaleva, N. E.; Shumyantseva, V. V.; Ivanov, A. S.; Archakov, A. I.

    2014-02-01

    The combination of SPR biosensor with electrochemical analysis was used for the study of protein-protein interaction between cytochromes CYP3A4 or 3А5 and cytochromes b5: the microsomal, mitochondrial forms of this protein, and 2 ≪chimeric≫ proteins. Kinetic constants of CYP3A4 and CYP3А5 complex formation with cytochromes b5 were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was observed upon their interactions with mitochondrial cytochrome b5. The electrochemical analysis of CYP3A4, CYP3A5, and cytochromes b5 immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 to -0.350 V (vs. Ag/AgCl).

  5. Upregulated mRNA expression of desaturase and elongase, two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish

    Enyu Yee-Ling

    2008-11-01

    Full Text Available Abstract Background Although unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3, docosahexaenoic acid (DHA, C22:6n-3 and arachidonic acid (ARA, C20:4n-6, collectively known as the highly unsaturated fatty acids (HUFA, play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6 and linolenic acid (LNA, C18:3n-3. As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6 and elongase (elovl5, involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed. Methods mRNA levels of fadsd6 and elovl5 in different ovarian follicle stages were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR assays. For analysis of the ovarian follicular fatty acid composition, gas chromatography was used. Results Our results have shown that desaturase displayed significant upregulation in expression during the oocyte maturation stage. Expression of elongase was significantly highest in pre-vitellogenic follicles, followed by maturation stage. Fatty acid composition analysis of different ovarian follicle stages also showed that ARA level was significantly highest in pre-vitellogenic and matured follicles. DHA level was highest in both late vitellogenic and maturation stage. Conclusion Collectively, our findings seem to suggest the existence of a HUFA synthesis system, which could be responsible for the synthesis of HUFA to promote oocyte maturation and possibly ovulation processes. The many advantages of zebrafish as model system to understand folliculogenesis will be

  6. Identification of Target Genes Involved in the Antiproliferative Effect of Enzyme-Modified Ginseng Extract in HepG2 Hepatocarcinoma Cell

    Sung-Il Jang

    2013-01-01

    Full Text Available Ginsenosides are ginseng saponins, which are the major biologically active components of Panax ginseng, often metabolized by intestinal bacteria into more effective forms. In this study, we found that the antiproliferative activity of ginseng increased after enzymatic processing of ginseng saponin (50% inhibitory concentration [IC50], >30 μg/mL, which may be the result of the accumulation of minor saponins, such as Rh1, Rg3, compound K, and PPT constituents in ginseng saponin. Using the Agilent PrimeView Human Gene Expression Array, we found that the expression of several genes involved in apoptosis (caspase-4, Annexin A2, HSPA9, AIFM1, UQCRC2, and caspase-7 were increased in HepG2 human hepatocarcinoma cells after their treatment with enzyme-modified ginseng extract (EMGE. Furthermore, several genes implicated in cell cycle progression (CDCA3, CDCA8, CABLES2, CDC25B, CNNM3, and CCNK showed decreased expression in HepG2 cells treated with EMGE. Finally, from flow cytometric analysis, we found that EMGE-treated HepG2 cells showed increased apoptotic sub-G1 population (24%, compared with that observed in DMSO-treated control cells (1.6%. Taken together, our results suggest that EMGE induces anticancer activity through the induction of apoptosis-related genes and cell cycle arrest via decreased expression of cell cycle regulatory genes.

  7. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    Doricakova, Aneta; Vrzal, Radim

    2015-11-01

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A. PMID:26341324

  8. Inhibition of CYP3A4 and CYP2C9 by podophyllotoxin: Implication for clinical drug–drug interactions

    Jin-Hui Song; Dong-Xue Sun; Bin Chen; Dai-Hong Ji; Jie Pu; Jie Xu; Feng-De Tian; Lin Guo

    2011-12-01

    Podophyllotoxin (PPT) and its derivatives exert significant anti-cancer activities, and one derivative etoposide is often utilized to treat various cancers in the clinic. The aim of the present study is to investigate the inhibitory effects of PPT on major cytochrome P450 (CYP) isoforms in human livers. Inhibition of CYP3A4, CYP2C9, CYP2C8, CYP2D6, CYP2E1 and CYP2A6 by PPT was investigated in the human liver microsomal system. Time-dependent inhibition of CYP3A4 by PPT was also evaluated. The results showed that PPT strongly exhibited inhibitory effects on CYP3A4 and CYP2C9 in a concentration-dependent manner. Half inhibition concentration (IC50) was 1.1±0.3 and 4.6±0.3 M for CYP3A4 and CYP2C9, respectively. Inhibition kinetic analysis showed that PPT exhibited competitive inhibition towards CYP3A4 and CYP2C9 with Ki of 1.6 and 2.0 M, respectively. Additionally, PPT exerted time-dependent inhibition towards CYP3A4 and the kinetic parameters were 4.4±2.1 M and 0.06±0.01 min–1 for KI and kinact, respectively. Our experimental data indicate that potential drug–drug interaction (DDI) might exist when PPT is co-administered with the substrates which mainly undergo CYP3A4- or CYP2C9-mediated metabolism.

  9. Monoester-Diterpene Aconitum Alkaloid Metabolism in Human Liver Microsomes: Predominant Role of CYP3A4 and CYP3A5

    Ling Ye

    2013-01-01

    Full Text Available Aconitum, widely used to treat rheumatoid arthritis for thousands of years, is a toxic herb that can frequently cause fatal cardiac poisoning. Aconitum toxicity could be decreased by properly hydrolyzing diester-diterpene alkaloids into monoester-diterpene alkaloids. Monoester-diterpene alkaloids, including benzoylaconine (BAC, benzoylmesaconine (BMA, and benzoylhypaconine (BHA, are the primary active and toxic constituents of processed Aconitum. Cytochrome P450 (CYP enzymes protect the human body by functioning as the defense line that limits the invasion of toxicants. Our purposes were to identify the CYP metabolites of BAC, BMA, and BHA in human liver microsomes and to distinguish which isozymes are responsible for their metabolism through the use of chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzyme. High-resolution mass spectrometry was used to characterize the metabolites. A total of 7, 8, and 9 metabolites were detected for BAC, BMA, and BHA, respectively. The main metabolic pathways were demethylation, dehydrogenation, demethylation-dehydrogenation, hydroxylation and didemethylation, which produced less toxic metabolites by decomposing the group responsible for the toxicity of the parent compound. Taken together, the results of the chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzymes experiments demonstrated that CYP3A4 and CYP3A5 have essential functions in the metabolism of BAC, BMA, and BHA.

  10. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6.

    Ke, A; Barter, Z; Rowland-Yeo, K; Almond, L

    2016-07-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug-drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0-1.7-fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz. PMID:27435752

  11. Contribution of CYP3A4 to catalysis of ketamine in human hepatic microsome%人肝微粒体中CYP3A4对氯胺酮代谢的催化作用

    赵芸慧; 田阿勇; 马虹; 王俊科

    2012-01-01

    目的 研究人肝脏微粒体中细胞色素P4503A4(CYP3A4)对氯胺酮代谢的催化作用.方法 用高效液相色谱法测定氯胺酮在人肝脏微粒体孵育液中的浓度变化,计算其代谢速率;分析该代谢速率与CYP3A4特异性底物硝苯地平代谢速率的相关性;并应用CYP3A4特异性抑制剂孕二烯酮检测CYP3A4对氯胺酮代谢的催化作用.结果 20例人肝脏微粒体中氯胺酮的代谢速率均值为(12.6±3.8)μmol·min-1·g-1 protein.该速率与CYP3A4活性探针硝苯地平代谢速率呈明显正相关(r=0.917,P<0.01).加入特异性抑制剂孕二烯酮组,氯胺酮的平均代谢速率明显低于正常孵育组,为(4.7±1.6)μmol·min-1·g-1 protein(P<0.01),抑制率为62.7%.结论 人肝微粒体中CYP3A4对氯胺酮代谢具有催化作用.%Aim To investigate the contribution of CYP3A4 to catalysis of ketamine in human hepatic mi-crosome. Methods The change of ketamine concentration in an incubation mixture with human hepatic microsomes was determined by high performance liquid chromatography ( HPLC) , and then the metabolic rate of ketamine was calculated. The correlation of the rate with rates of metabolism of CYP3A4 selective substrate nifedipine, and the effect of CYP3A4 specific inhibitor gestodene on ketamine metabolism were examined. Results The metabolic rate of ketamine in the twenty ca -ses of microsomes was ( 12. 6 ± 3. 8 μmol · min-1 ·g-1 protein) on average. The average rate of ketamine metabolism showed obvious positive correlation to that of nifedipine (activity probe of CYP3A4) (r =0. 917, P < 0. 01). After addition of gestodene (the specific inhibitor of CYP3A4) , the average metabolic rate of ketamine (4.7 ±1.6 μmol· min-1 · g-1 protein) was slower than that without gestodene (P < 0. 01). The inhibition degree was 62.7%. Conclusion CYP3A4 is responsible for metabolism of ketamine in human he -patic microsome.

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

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

    2006-05-24

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

  13. The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity.

    Wu, Qiangen; Ning, Baitang; Xuan, Jiekun; Ren, Zhen; Guo, Lei; Bryant, Matthew S

    2016-06-24

    Amiodarone is a widely used potent antiarrhythmic for the treatment of cardiac disease; however, its use is often discontinued due to numerous adverse effects, including hepatotoxicity. To investigate the role of drug metabolism in this liver toxicity, amiodarone and its major metabolite desethylamiodarone were incubated with HepG2 cells overexpressing a series of cytochrome P450 (CYP) isoforms. Significantly higher cytotoxicity of amiodarone was observed in HepG2 cells overexpressing CYP3A4 or CYP1A1, compared with that observed in empty vector transduced control cells. Further, higher levels of the more potent hepatotoxic metabolite desethylamiodarone were detected in CYP3A4 or CYP1A1 expressed cells. The CYP3A4 inhibitor ketoconazole and the CYP1A1 inhibitor α-naphthoflavone drastically inhibited the metabolism of amiodarone to desethylamiodarone. Along with the inhibition of CYP1A1 or CYP3A4, the cytotoxicity of amiodarone was significantly reduced. These data indicate that the metabolism of amiodarone to desethylamiodarone by CYP1A1 or CYP3A4 plays an important role in the hepatocellular toxicity of amiodarone. PMID:27113703

  14. Losartan attenuates chronic cigarette smoke exposure-induced pulmonary arterial hypertension in rats: Possible involvement of angiotensin-converting enzyme-2

    Chronic cigarette smoking induces pulmonary arterial hypertension (PAH) by largely unknown mechanisms. Renin-angiotensin system (RAS) is known to function in the development of PAH. Losartan, a specific angiotensin II receptor antagonist, is a well-known antihypertensive drug with a potential role in regulating angiotensin-converting enzyme-2 (ACE2), a recently found regulator of RAS. To determine the effect of losartan on smoke-induced PAH and its possible mechanism, rats were daily exposed to cigarette smoke for 6 months in the absence and in the presence of losartan. Elevated right ventricular systolic pressure (RVSP), thickened wall of pulmonary arteries with apparent medial hypertrophy along with increased angiotensin II (Ang II) and decreased ACE2 levels were observed in smoke-exposed-only rats. Losartan administration ameliorated pulmonary vascular remodeling, inhibited the smoke-induced RVSP and Ang II elevation and partially reversed the ACE2 decrease in rat lungs. In cultured primary pulmonary artery smooth muscle cells (PASMCs) from 3- and 6-month smoke-exposed rats, ACE2 levels were significantly lower than in those from the control rats. Moreover, PASMCs from 6-month exposed rats proliferated more rapidly than those from 3-month exposed or control rats, and cells grew even more rapidly in the presence of DX600, an ACE2 inhibitor. Consistent with the in vivo study, in vitro losartan pretreatment also inhibited cigarette smoke extract (CSE)-induced cell proliferation and ACE2 reduction in rat PASMCs. The results suggest that losartan may be therapeutically useful in the chronic smoking-induced pulmonary vascular remodeling and PAH and ACE2 may be involved as part of its mechanism. Our study might provide insight into the development of new therapeutic interventions for PAH smokers.

  15. Molecular and enzymatic characterization of two enzymes BmPCD and BmDHPR involving in the regeneration pathway of tetrahydrobiopterin from the silkworm Bombyx mori.

    Li, Wentian; Gong, Meixia; Shu, Rui; Li, Xin; Gao, Junshan; Meng, Yan

    2015-08-01

    Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthase so that BH4 plays a key role in many biological processes. BH4 deficiency is associated with numerous metabolic syndromes and neuropsychological disorders. BH4 concentration in mammals is maintained through a de novo synthesis pathway and a regeneration pathway. Previous studies showed that the de novo pathway of BH4 is similar between insects and mammals. However, knowledge about the regeneration pathway of BH4 (RPB) is very limited in insects. Several mutants in the silkworm Bombyx mori have been approved to be associated with BH4 deficiency, which are good models to research on the RPB in insects. In this study, homologous genes encoding two enzymes, pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) involving in RPB have been cloned and identified from B. mori. Enzymatic activity of DHPR was found in the fat body of wild type silkworm larvae. Together with the transcription profiles, it was indicated that BmPcd and BmDhpr might normally act in the RPB of B. mori and the expression of BmDhpr was activated in the brain and sexual glands while BmPcd was expressed in a wider special pattern when the de novo pathway of BH4 was lacked in lemon. Biochemical analyses showed that the recombinant BmDHPR exhibited high enzymatic activity and more suitable parameters to the coenzyme of NADH in vitro. The results in this report give new information about the RPB in B. mori and help in better understanding insect BH4 biosynthetic networks. PMID:25899859

  16. Evaluation of a SUMO E2 conjugating enzyme involved in resistance to Clavibacter michiganensis subsp. michiganensis in Solanum peruvianum, through a tomato mottle virus VIGS assay

    Mayra Janeth Esparza-Araiza

    2015-12-01

    Full Text Available Clavibacter michiganensis subsp. michiganensis (Cmm causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianum. Previous research showed up-regulation of a SUMO E2 conjugating enzyme (SCEI transcript in resistant S. peruvianum compared to susceptible S. lycopersicum following infection by Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of the gene from S. peruvianum was cloned into a novel virus-induced gene-silencing (VIGS vector based on the geminivirus Tomato Mottle Virus (ToMoV. Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, which resulted in leaf bleaching. The ToMoV_SCEI construct resulted in approx. 61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. VIGS of SCEI in S. peruvianum resulted in unilateral wilting (15 dpi and subsequent death (20 dpi of the entire plant after Cmm inoculation, whereas empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. SCEI-silenced plants also showed higher Cmm colonization with an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of WRKY transcription factors, which may lead to expression of proteins involved in salicylic acid-dependent defense responses.

  17. Evaluation of a SUMO E2 Conjugating Enzyme Involved in Resistance to Clavibacter michiganensis Subsp. michiganensis in Solanum peruvianum, Through a Tomato Mottle Virus VIGS Assay.

    Esparza-Araiza, Mayra J; Bañuelos-Hernández, Bernardo; Argüello-Astorga, Gerardo R; Lara-Ávila, José P; Goodwin, Paul H; Isordia-Jasso, María I; Castillo-Collazo, Rosalba; Rougon-Cardoso, Alejandra; Alpuche-Solís, Ángel G

    2015-01-01

    Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianum. Previous research showed up-regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in S. peruvianum compared to S. lycopersicum following infection with Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of SCEI from S. peruvianum was cloned into a novel virus-induced gene-silencing (VIGS) vector based on the geminivirus, Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, resulting in leaf bleaching. VIGS with the ToMoV_SCEI construct resulted in ~61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. The SCEI-silenced plants showed unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas the empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. The SCEI-silenced plants showed higher Cmm colonization and an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of transcription factors, leading to expression of proteins involved in salicylic acid-dependent defense responses. PMID:26734014

  18. Association of CYP3A4/5, ABCB1 and ABCC2 polymorphisms and clinical outcomes of Thai breast cancer patients treated with tamoxifen

    Sensorn I

    2013-08-01

    Full Text Available Insee Sensorn,1 Ekaphop Sirachainan,2 Montri Chamnanphon,3 Ekawat Pasomsub,4 Narumol Trachu,5 Porntip Supavilai,1 Chonlaphat Sukasem,3 Darawan Pinthong11Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand; 2Division of Medical Oncology, Department of Medicine, 3Division for Pharmacogenomics and Personalized Medicine, 4Division for Virology, Department of Pathology, 5Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, ThailandBackground: Pharmacogenetic study of cytochrome P450 (CYP gene CYP2D6 and tamoxifen outcomes remain controversial. Apart from CYP2D6, other drug-metabolizing enzymes and transporters also play a role in tamoxifen metabolic pathways. The aim of this study is to investigate the impact of CYP3A4/5, ABCB1, and ABCC2 polymorphisms on the risk of recurrence in Thai patients who received tamoxifen adjuvant therapy.Methods: Patients with early-stage breast cancer who received tamoxifen adjuvant therapy were recruited in this study. All six single-nucleotide polymorphisms (SNPs, including CYP3A4*1B (-392 A>G/*18(878 T>C, CYP3A5*3(6986 G>A, ABCB1 3435 C>T, ABCC2*1C (-24 C>T, and ABCC2 68231 A>G, were genotyped using real-time polymerase chain reaction assays. The impacts of genetic variants on disease-free survival (DFS were analyzed using the Kaplan–Meier method and Cox regression analysis.Results: The ABCB1 3435 C>T was found to have the highest allele frequency among other variants; however, CYP3A4*1B/*18 could not be found in this study. Patients with heterozygous ABCB1 3435 CT genotype showed significantly shorter DFS than those with homozygous 3435 CC genotype (P = 0.041. In contrast, patients who carried homozygous 3435 TT genotype showed no difference in DFS from wild-type 3435 CC patients. Cox regression analysis showed that the relative risk of recurrence was increased by five times (P = 0.043; hazard ratio = 5.11; 95% confidence interval: 1.05–24

  19. Cytochrome P450 3A4*22, PPAR-α, and ARNT polymorphisms and clopidogrel response

    Kreutz RP

    2013-12-01

    Full Text Available Rolf P Kreutz,1,2 Janelle Owens,2 Yan Jin,2 Perry Nystrom,2 Zeruesenay Desta,2 Yvonne Kreutz,2 Jeffrey A Breall,1 Lang Li,3 ChienWei Chiang,3 Richard J Kovacs,1 David A Flockhart21Krannert Institute of Cardiology, 2Division of Clinical Pharmacology, Indiana University School of Medicine, 3Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, Indiana, USAAbstract: Recent candidate gene studies using a human liver bank and in vivo validation in healthy volunteers identified polymorphisms in cytochrome P450 (CYP 3A4 gene (CYP3A4*22, Ah-receptor nuclear translocator (ARNT, and peroxisome proliferator-activated receptor-α (PPAR-α genes that are associated with the CYP3A4 phenotype. We hypothesized that the variants identified in these genes may be associated with altered clopidogrel response, since generation of clopidogrel active metabolite is, partially mediated by CYP3A activity. Blood samples from 211 subjects, of mixed racial background, with established coronary artery disease, who had received clopidogrel, were analyzed. Platelet aggregation was determined using light transmittance aggregometry (LTA. Genotyping for CYP2C19*2, CYP3A4*22, PPAR-α (rs4253728, rs4823613, and ARNT (rs2134688 variant alleles was performed using Taqman® assays. CYP2C19*2 genotype was associated with increased on-treatment platelet aggregation (adenosine diphosphate 20 µM; P=0.025. No significant difference in on-treatment platelet aggregation, as measured by LTA during therapy with clopidogrel, was demonstrated among the different genotypes of CYP3A4*22, PPAR-α, and ARNT. These findings suggest that clopidogrel platelet inhibition is not influenced by the genetic variants that have previously been associated with reduced CYP3A4 activity.Keywords: clopidogrel, pharmacogenetics, CYP450, platelet aggregation

  20. Pharmacokinetic variability of clarithromycin and differences in CYP3A4 activity in patients with cystic fibrosis

    Dalbøge, C S; Nielsen, X C; Dalhoff, K;

    2014-01-01

    3A4-activity and clarithromycin metabolism was demonstrated (P < 0.05). CONCLUSION: The large variation in the clarithromycin pharmacokinetics in cystic fibrosis patients may cause treatment failure. The Erythromycin Breath Test could be valuable in identifying cystic fibrosis patients in risk of...... treatment failure/drug toxicity....

  1. Overexpression of CYP3A4 in a COLO 205 Colon Cancer Stem Cell Model in vitro

    Cancer stem cells (CSCs) seem to constitute a subpopulation of tumor cells that escape from chemotherapy and cause recurrent disease. Low proliferation rates, protection in a stem cell niche and overexpression of drug resistance proteins are considered to confer chemoresistance. We established an in vitro colon CSC-like model using the COLO 205 cell line, which revealed transiently increased expression of CD133 when transferred to serum-free stem cell culture medium. Assessment of global gene expression of COLO 205 cells under these conditions identified a set of upregulated genes including cytochrome P450 3A4 (CYP3A4) and aldehyde dehydrogenase 1A1 (ALDH1A1), as confirmed by real-time qPCR. ALDH1A1 is a CSC marker for certain tumor entities and confers resistance to cyclophosphamide. CYP3A4 is expressed in liver and colon and its overexpression seems particularly relevant in colon cancer, since it inactivates irinotecan and other xenobiotics, such as taxols and vinca alkaloids. In conclusion, this COLO 205 model provides evidence for CD133 induction concomitant with overexpression of CYP3A4, which, together with ATP-binding cassette, subfamily G, member 2 (ABCG2) and others, may have a role in chemoresistant colon CSCs and a negative impact on disease-free survival in colon cancer patients

  2. Overexpression of CYP3A4 in a COLO 205 Colon Cancer Stem Cell Model in vitro

    Olszewski, Ulrike [Ludwig Boltzmann Cluster of Translational Oncology, c/o Balderichgasse 26/13, A-1170 Vienna (Austria); Liedauer, Richard [Department of Pathophysiology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna (Austria); Ausch, Christoph [Department of Surgery, Danube Hospital, A-1220 Vienna (Austria); Thalhammer, Theresia [Department of Pathophysiology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna (Austria); Hamilton, Gerhard, E-mail: gerhard.hamilton@toc.lbg.ac.at [Ludwig Boltzmann Cluster of Translational Oncology, c/o Balderichgasse 26/13, A-1170 Vienna (Austria)

    2011-03-22

    Cancer stem cells (CSCs) seem to constitute a subpopulation of tumor cells that escape from chemotherapy and cause recurrent disease. Low proliferation rates, protection in a stem cell niche and overexpression of drug resistance proteins are considered to confer chemoresistance. We established an in vitro colon CSC-like model using the COLO 205 cell line, which revealed transiently increased expression of CD133 when transferred to serum-free stem cell culture medium. Assessment of global gene expression of COLO 205 cells under these conditions identified a set of upregulated genes including cytochrome P450 3A4 (CYP3A4) and aldehyde dehydrogenase 1A1 (ALDH1A1), as confirmed by real-time qPCR. ALDH1A1 is a CSC marker for certain tumor entities and confers resistance to cyclophosphamide. CYP3A4 is expressed in liver and colon and its overexpression seems particularly relevant in colon cancer, since it inactivates irinotecan and other xenobiotics, such as taxols and vinca alkaloids. In conclusion, this COLO 205 model provides evidence for CD133 induction concomitant with overexpression of CYP3A4, which, together with ATP-binding cassette, subfamily G, member 2 (ABCG2) and others, may have a role in chemoresistant colon CSCs and a negative impact on disease-free survival in colon cancer patients.

  3. N-Hydroxylation of dapsone by multiple enzymes of cytochrome P450: implications for inhibition of haemotoxicity.

    Gill, H J; Tingle, M D; Park, B.K.

    1995-01-01

    1. The adverse reactions associated with the administration of dapsone are believed to be caused by metabolism to its hydroxylamine. Previous reports suggest that CYP3A4 is responsible for this biotransformation [1]. 2. Data presented in this paper illustrate the involvement of more than one cytochrome P450 enzyme in dapsone hydroxylamine formation using human liver microsomes. Eadie-Hofstee plots demonstrated bi-phasic kinetics in several livers. No correlation could be established between h...

  4. Association of polymorphisms in CYP19A1 and CYP3A4 genes with lower urinary tract symptoms, prostate volume, uroflow and PSA in a population-based sample

    R. Berges; A. Gsur; E. Feik; K. Höfner; T. Senge; L. Pientka; A. Baierl; M.C. Michel; A. Ponholzer; S. Madersbacher

    2011-01-01

    PURPOSE: The known importance of testosterone for the development of benign prostatic hyperplasia (BPH) prompted us to test the hypothesis whether polymorphisms of two genes (CYP19A1 and CYP3A4) involved in testosterone metabolism are associated with clinical BPH-parameters. METHODS: A random sample

  5. An enzyme activity in normal and ataxia telangiectasia cell lines which is involved in the repair of γ-irradiation-induced DNA damage

    An enzyme that enhances the activity of DNA polymerase I (EC 2.7.7.7) for γ-irradiated calf thymus DNA was demonstrated in cellular extracts of normal human fibroblasts and lymphoid-cell lines. This enzyme was found to be deficient in all cellular extracts of fibroblasts and lymphoid-cell lines examined from patients with the autosomal recessive disease ataxia telangiectasia. The activity in cellular extracts from normal fibroblasts was removed when heated to 1000C for 2 min or when the assay was performed at 40C. No significant deficiency in primer activating enzyme activity was observed in cell-free extracts of lymphoid lines from patients with xeroderma pigmentosum, Huntington's chorea or neurofibromatosis, or from an ataxia telangiectasia heterozygote. (author)

  6. 二甲亚砜对细胞色素P450酶3A4和2C9的影响%Effects of Dimethyl Sulfoxide on Cytochrome P450 3A4 and 2C9

    王彧杰; 王媛媛; 王蓉; 原永芳

    2014-01-01

    Objective To investigate effects of 0.01%, 0.05%, 0.1% dimethyl sulfoxide (DMSO) on expression of CYP3A4 and CYP2C9 mRNA and protein. Methods Chang liver cells were divided into control group (dealed with RPMI-1640 singly), DMSO groups (dealed with 0.01%, 0.05%, 0.1%DMSO), testosterone groups (dealed with 1, 10, 100μmol/L testosterone), rifampicin groups (dealed with 1, 10, 100μmol/L Rifampicin), DMSO+testosterone groups (0.01%, 0.05%, 0.1%DMSO+10μmol/L testosterone) and DMSO+rifampicin groups (dealed with 0.01%, 0.05%, 0.1%DMSO+10μmol/L Rifampicin). The expression of CYP3A4 and CYP2C9 mRNA were detected by RT-qPCR, and the expression of CYP3A4 and CYP2C9 protein were detected by Western blot. Results Compared with control group, the expression of CYP3A4 mRNA and protein was increased by 0.1%DMSO (P0.05). Compared with control group, there was no statistically significant difference on the expression of CYP2C9 mRNA and protein in DMSO groups (P> 0.05). Compared with control group, there was no statistically significant difference on the expres-sion of CYP3A4 mRNA in DMSO+testosterone groups (P>0.05). Compared with testosterone group, the expression of CYP3A4 protein was induced in 0.01%, 0.05%, 0.1%DMSO + testosterone group. Compared with rifampicin group, the expression of CYP2C9 protein was increased in 0.01%, 0.05%, 0.1% DMSO+Rifampicin group, but no statistically sig-nificant difference on CYP2C9 mRNA (P > 0.05). Conclusion The efficiency of 0.01%, 0.05% and 0.1% DMSO is greater on CYP3A4 than CYP2C9 in vitro test. 0.01%, 0.05%, 0.1% DMSO can influence expression of CYP3A4, but have poor effect on expression of CYP2C9 in vitro. DMSO as solvent can influence results of experiment.%目的:研究0.01%、0.05%、0.1%二甲亚砜(DMSO)对细胞色素P450(CYP450)酶系中3A4、2C9两个亚型基因及蛋白表达水平的影响。方法 Chang肝脏细胞经处理后,分为空白对照组(仅含培养液)、DMSO组(分别采用0.01%、0.05%、0.1%DMSO

  7. Human variation and CYP enzyme contribution in benfuracarb metabolism in human in vitro hepatic models.

    Abass, Khaled; Reponen, Petri; Mattila, Sampo; Rautio, Arja; Pelkonen, Olavi

    2014-01-13

    Human responses to the toxicological effects of chemicals are often complicated by a substantial interindividual variability in toxicokinetics, of which metabolism is often the most important factor. Therefore, we investigated human variation and the contributions of human-CYP isoforms to in vitro metabolism of benfuracarb. The primary metabolic pathways were the initial sulfur oxidation to benfuracarb-sulfoxide and the nitrogen-sulfur bond cleavage to carbofuran (activation). The Km, Vmax, and CL(int) values of carbofuran production in ten individual hepatic samples varied 7.3-, 3.4-, and 5.4-fold, respectively. CYP2C9 and CYP2C19 catalyzed benfuracarb sulphur oxidation. Carbofuran formation, representing from 79% to 98% of the total metabolism, was catalyzed predominantly by CYP3A4. The calculated relative contribution of CYP3A4 to carbofuran formation was 93%, while it was 4.4% for CYP2C9. The major contribution of CYP3A4 in benfuracarb metabolism was further substantiated by showing a strong correlation with CYP3A4-selective markers midazolam-1'-hydroxylation and omeprazole-sulfoxidation (r=0.885 and 0.772, respectively). Carbofuran formation was highly inhibited by the CYP3A inhibitor ketoconazole. Moreover, CYP3A4 marker activities were relatively inhibited by benfuracarb. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro activation of benfuracarb and that CYP3A4-catalyzed metabolism is the primary source of interindividual differences. PMID:24016712

  8. Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters.

    Vermeer, Lydia M M; Isringhausen, Caleb D; Ogilvie, Brian W; Buckley, David B

    2016-03-01

    Ketoconazole is a potent CYP3A4/5 inhibitor and, until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency as a strong CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and European Medicines Agency recommended suspension of ketoconazole use in DDI studies in 2013. The FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of drug transporter activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir, and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto-, and N-desalkyl itraconazole) toward 13 drug transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, P-gp, BCRP, MRP2, MRP3, and BSEP) were systematically assessed in transporter-expressing HEK-293 cell lines or membrane vesicles. In vitro findings were translated into clinical context with the basic static model approaches outlined by the FDA in its 2012 draft guidance on DDIs. The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin, and itraconazole each have unique transporter inhibition profiles. None of the alternatives to ketoconazole provided a clean inhibition profile toward the 13 drug transporters evaluated. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when transporters are potentially involved in a victim drug's pharmacokinetics. PMID:26668209

  9. Evidence for a repair enzyme complex involving ERCC1, and the correcting activities of ERCC4, ERCC11 and the xeroderma pigmentosum group F.

    A.J. van Vuuren (Hanneke); E. Appeldoorn (Esther); H. Odijk (Hanny); A. Yasui (Akira); N.G.J. Jaspers (Nicolaas); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1993-01-01

    textabstractNucleotide excision repair (NER), one of the major cellular DNA repair systems, removes a wide range of lesions in a multi-enzyme reaction. In man, a NER defect due to a mutation in one of at least 11 distinct genes, can give rise to the inherited repair disorders xeroderma pigmentosum (

  10. Screening for and Identification of Novel Glucarpidase Producing Bacteria : Cloning and molecular characterisation of novel enzymes involved in ADEPT for cancer treatment

    Alqahtani, Alanood; Alyafei, Afrah; Abdallah, Fatma; Latiff, Aishah; Groves, Matthew; Dömling, Alex; Goda, Sayed

    2014-01-01

    Antibody Directed Enzyme Prodrug Therapy (ADEPT) is a novel therapy which has already been implemented in cancer therapy to solve the problem of drug resistance and lack of tumor selectivity. Repeated cycles of (ADEPT) and the use of glucarpidase in detoxification of cytotoxic methotrexate (MTX) are

  11. Phosphoenolpyruvate carboxylase, NADP-malic enzyme, and pyruvate, phosphate dikinase are involved in the acclimation of Nicotiana tabacum L. to drought stress

    Doubnerová-Hýsková, V.; Miedzińska, L.; Dobrá, Jana; Vaňková, Radomíra; Ryšlavá, H.

    2014-01-01

    Roč. 171, č. 5 (2014), s. 19-25. ISSN 0176-1617 R&D Projects: GA MŠk 1M0505 Institutional support: RVO:61389030 Keywords : Drought * NADP-malic enzyme * Nicotiana tabacum L. Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.557, year: 2014

  12. Drug membrane transporters and CYP3A4 are affected by hypericin, hyperforin or aristoforin in colon adenocarcinoma cells.

    Šemeláková, M; Jendželovský, R; Fedoročko, P

    2016-07-01

    Our previous results have shown that the combination of hypericin-mediated photodynamic therapy (HY-PDT) at sub-optimal dose with hyperforin (HP) (compounds of Hypericum sp.), or its stable derivative aristoforin (AR) stimulates generation of reactive oxygen species (ROS) leading to antitumour activity. This enhanced oxidative stress evoked the need for an explanation for HY accumulation in colon cancer cells pretreated with HP or AR. Generally, the therapeutic efficacy of chemotherapeutics is limited by drug resistance related to the overexpression of drug efflux transporters in tumour cells. Therefore, the impact of non-activated hypericin (HY), HY-PDT, HP and AR on cell membrane transporter systems (Multidrug resistance-associated protein 1-MRP1/ABCC1, Multidrug resistance-associated protein 2-MRP2/ABCC2, Breast cancer resistance protein - BCRP/ABCG2, P-glycoprotein-P-gp/ABCC1) and cytochrome P450 3A4 (CYP3A4) was evaluated. The different effects of the three compounds on their expression, protein level and activity was determined under specific PDT light (T0+, T6+) or dark conditions (T0- T6-). We found that HP or AR treatment affected the protein levels of MRP2 and P-gp, whereas HP decreased MRP2 and P-gp expression mostly in the T0+ and T6+ conditions, while AR decreased MRP2 in T0- and T6+. Moreover, HY-PDT treatment induced the expression of MRP1. Our data demonstrate that HP or AR treatment in light or dark PDT conditions had an inhibitory effect on the activity of individual membrane transport proteins and significantly decreased CYP3A4 activity in HT-29 cells. We found that HP or AR significantly affected intracellular accumulation of HY in HT-29 colon adenocarcinoma cells. These results suggest that HY, HP and AR might affect the efficiency of anti-cancer drugs, through interaction with membrane transporters and CYP3A4. PMID:27261575

  13. Modulation of CYP3A4 activity alters the cytotoxicity of lipophilic phycotoxins in human hepatic HepaRG cells.

    Ferron, P J; Hogeveen, K; De Sousa, G; Rahmani, R; Dubreil, E; Fessard, V; Le Hegarat, L

    2016-06-01

    The aim of this study was to investigate (i) the cytotoxic effects of lipophilic phycotoxins, including okadaic acid (OA) and dinophysistoxin-1 and -2 (DTX-1 and DTX-2), pectenotoxin-2 (PTX-2), yessotoxin (YTX), spirolide (SPX), and azaspiracids-1, -2 and -3 (AZA-1, AZA-2 and AZA-3), in human HepaRG cells using a multiparametric high content analysis approach, (ii) the ability of nine lipophilic phycotoxins to act as PXR agonists in a HepG2-PXR cell line, (iii) their potential to induce CYP450 activity, and (iv) the role of CYP3A4 in cytotoxicity induced by lipophilic phycotoxins. Our results indicate that while OA, DTX-1 and DTX-2 activated PXR-dependent transcriptional activity in HepG2 cells, no increase of CYP450 (1A2, 3A4, 2C9, 2C19) activities were observed in HepaRG cell following a 72h treatment with these toxins. Multiparametric analysis showed that OA, DTX-1, DTX-2, and PTX-2 were highly cytotoxic in HepaRG cells; inducing cell loss, activation of caspase-3 and γ-H2AX formation. However, no toxicity was observed for YTX, SPX, and AZAs. Moreover, we found that inhibition of CYP3A4 activity by ketoconazole enhances the toxic effects of OA, DTX-1, DTX-2, and PTX-2 in HepaRG cells. Taken together, these results suggest that CYP3A4-mediated metabolism of some lipophilic phycotoxins decreases their in vitro toxicity. PMID:26956883

  14. Crystal Structure and Mechanism of Tryptophan 2,3-Dioxygenase, a Heme Enzyme Involved in Tryptophan Catabolism and in Quinolinate Biosynthesis

    Zhang,Y.; Kang, S.; Mukherjee, T.; Bale, S.; Crane, B.; Begley, T.; Ealick, S.

    2007-01-01

    The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 {angstrom}. TDO catalyzes the irreversible oxidation of L-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate L-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.

  15. Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes.

    Anderson, K L; Salyers, A A

    1989-01-01

    Bacteroides thetaiotaomicron can utilize amylose, amylopectin, and pullulan as sole sources of carbon and energy. The enzymes that degrade these polysaccharides were found to be primarily cell associated rather than extracellular. Although some activity was detected in extracellular fluid, this appeared to be the result of cell lysis. The cell-associated amylase, amylopectinase, and pullulanase activities partitioned similarly to the periplasmic marker, acid phosphatase, when cells were expos...

  16. Enhancement of the tolerance to oxidative stress in cucumber (Cucumis sativus L.) seedlings by UV-B irradiation: Possible involvement of phenolic compounds and antioxidative enzymes

    L.) seedlings were irradiated or not irradiated with UV-B for several days in environment-controlled growth chambers. The first leaves irradiated with UV-B were retarded in growth but simultaneously acquired a remarkably high tolerance to oxidative stress, as induced by paraquat treatment, compared with the non-irradiated leaves. This enhanced tolerance was observed within 1d after the start of UV-B irradiation and was maintained during the 12 d period of UV-B treatment. The effects of UV-B on several antioxidative enzymes were examined, and activities of superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase, but not of glutathione reductase, were found to be enhanced. However, activation of these enzymes occurred only from 6 d after the start of irradiation. In contrast, accumulation of phenolic compounds was observed within 1d after the start of UV-B irradiation. HPLC analysis of phenolic compounds showed the distinct enhancement of a substance, which may have antioxidative properties in cucumber seedlings irradiated with UV-B. On the basis of these results, we conclude that not only antioxidative enzymes but also other factors in cucumber seedlings irradiated with UV-B, such as phenolic compounds, may participate in the enhanced tolerance to oxidative stress

  17. Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains.

    Valk, Vincent; Eeuwema, Wieger; Sarian, Fean D; van der Kaaij, Rachel M; Dijkhuizen, Lubbert

    2015-10-01

    The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation. PMID:26187958

  18. Differential induction of enzymes and genes involved in lipid metabolism in liver and visceral adipose tissue of juvenile yellow catfish Pelteobagrus fulvidraco exposed to copper

    Highlights: •Cu downregulates lipogenesis and reduces lipid deposition in liver and adipose tissue. •Mechanism of Cu affecting lipid metabolism is determined at the enzymatic and molecular levels. •Cu exposure differentially influences lipid metabolism between liver and adipose tissue. -- Abstract: The present study was conducted to determine the mechanism of waterborne Cu exposure influencing lipid metabolism in liver and visceral adipose tissue (VAT) of juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were exposed to four waterborne copper (Cu) concentrations (2 (control), 24 (low), 71 (medium), 198 (high) μg Cu/l, respectively) for 6 weeks. Waterborne Cu exposure had a negative effect on growth and several condition indices (condition factor, viscerosomatic index, hepatosomatic index and visceral adipose index). In liver, lipid content, activities of lipogenic enzymes (6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and fatty acid synthase (FAS)) as well as mRNA levels of 6PGD, G6PD, FAS and sterol-regulator element-binding protein-1 (SREBP-1) genes decreased with increasing Cu concentrations. However, activity and mRNA level of lipoprotein lipase (LPL) gene in liver increased. In VAT, G6PD, ME and LPL activities as well as the mRNA levels of FAS, LPL and PPARγ genes decreased in fish exposed to higher Cu concentrations. The differential Pearson correlations between transcription factors (SREBP-1 and peroxisome proliferators-activated receptor-γ (PPARγ)), and the activities and mRNA expression of lipogenic enzymes and their genes were observed between liver and VAT. Thus, our study indicated that reduced lipid contents in liver and VAT after Cu exposure were attributable to the reduced activities and mRNA expression of lipogenic enzymes and their genes in these tissues. Different response patterns of several tested enzymes and genes to waterborne Cu

  19. Differential induction of enzymes and genes involved in lipid metabolism in liver and visceral adipose tissue of juvenile yellow catfish Pelteobagrus fulvidraco exposed to copper

    Chen, Qi-Liang; Luo, Zhi, E-mail: luozhi99@yahoo.com.cn; Pan, Ya-Xiong; Zheng, Jia-Lang; Zhu, Qing-Ling; Sun, Lin-Dan; Zhuo, Mei-Qin; Hu, Wei

    2013-07-15

    Highlights: •Cu downregulates lipogenesis and reduces lipid deposition in liver and adipose tissue. •Mechanism of Cu affecting lipid metabolism is determined at the enzymatic and molecular levels. •Cu exposure differentially influences lipid metabolism between liver and adipose tissue. -- Abstract: The present study was conducted to determine the mechanism of waterborne Cu exposure influencing lipid metabolism in liver and visceral adipose tissue (VAT) of juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were exposed to four waterborne copper (Cu) concentrations (2 (control), 24 (low), 71 (medium), 198 (high) μg Cu/l, respectively) for 6 weeks. Waterborne Cu exposure had a negative effect on growth and several condition indices (condition factor, viscerosomatic index, hepatosomatic index and visceral adipose index). In liver, lipid content, activities of lipogenic enzymes (6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and fatty acid synthase (FAS)) as well as mRNA levels of 6PGD, G6PD, FAS and sterol-regulator element-binding protein-1 (SREBP-1) genes decreased with increasing Cu concentrations. However, activity and mRNA level of lipoprotein lipase (LPL) gene in liver increased. In VAT, G6PD, ME and LPL activities as well as the mRNA levels of FAS, LPL and PPARγ genes decreased in fish exposed to higher Cu concentrations. The differential Pearson correlations between transcription factors (SREBP-1 and peroxisome proliferators-activated receptor-γ (PPARγ)), and the activities and mRNA expression of lipogenic enzymes and their genes were observed between liver and VAT. Thus, our study indicated that reduced lipid contents in liver and VAT after Cu exposure were attributable to the reduced activities and mRNA expression of lipogenic enzymes and their genes in these tissues. Different response patterns of several tested enzymes and genes to waterborne Cu

  20. Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells

    Pregnane X receptor (PXR) is a key transcription factor that regulates drug metabolizing enzymes such as cytochrome P450 (CYP) 3A4, and plays important roles in intestinal first-pass metabolism. Although there is a large inter-individual heterogeneity with intestinal CYP3A4 expression and activity, the mechanism driving these differences is not sufficiently explained by genetic variability of PXR or CYP3A4. We examined whether epigenetic mechanisms are involved in the regulation of PXR/CYP3A4 pathways in colon cancer cells. mRNA levels of PXR, CYP3A4 and vitamin D receptor (VDR) were evaluated by quantitative real-time PCR on 6 colon cancer cell lines (Caco-2, HT29, HCT116, SW48, LS180, and LoVo). DNA methylation status was also examined by bisulfite sequencing of the 6 cell lines and 18 colorectal cancer tissue samples. DNA methylation was reversed by the treatment of these cell lines with 5-aza-2'-deoxycytidine (5-aza-dC). The 6 colon cancer cell lines were classified into two groups (high or low expression cells) based on the basal level of PXR/CYP3A4 mRNA. DNA methylation of the CpG-rich sequence of the PXR promoter was more densely detected in the low expression cells (Caco-2, HT29, HCT116, and SW48) than in the high expression cells (LS180 and LoVo). This methylation was reversed by treatment with 5-aza-dC, in association with re-expression of PXR and CYP3A4 mRNA, but not VDR mRNA. Therefore, PXR transcription was silenced by promoter methylation in the low expression cells, which most likely led to downregulation of CYP3A4 transactivation. Moreover, a lower level of PXR promoter methylation was observed in colorectal cancer tissues compared with adjacent normal mucosa, suggesting upregulation of the PXR/CYP3A4 mRNAs during carcinogenesis. PXR promoter methylation is involved in the regulation of intestinal PXR and CYP3A4 mRNA expression and might be associated with the inter-individual variability of the drug responses of colon cancer cells

  1. Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells

    Otsuka Koki

    2011-02-01

    Full Text Available Abstract Background Pregnane X receptor (PXR is a key transcription factor that regulates drug metabolizing enzymes such as cytochrome P450 (CYP 3A4, and plays important roles in intestinal first-pass metabolism. Although there is a large inter-individual heterogeneity with intestinal CYP3A4 expression and activity, the mechanism driving these differences is not sufficiently explained by genetic variability of PXR or CYP3A4. We examined whether epigenetic mechanisms are involved in the regulation of PXR/CYP3A4 pathways in colon cancer cells. Methods mRNA levels of PXR, CYP3A4 and vitamin D receptor (VDR were evaluated by quantitative real-time PCR on 6 colon cancer cell lines (Caco-2, HT29, HCT116, SW48, LS180, and LoVo. DNA methylation status was also examined by bisulfite sequencing of the 6 cell lines and 18 colorectal cancer tissue samples. DNA methylation was reversed by the treatment of these cell lines with 5-aza-2'-deoxycytidine (5-aza-dC. Results The 6 colon cancer cell lines were classified into two groups (high or low expression cells based on the basal level of PXR/CYP3A4 mRNA. DNA methylation of the CpG-rich sequence of the PXR promoter was more densely detected in the low expression cells (Caco-2, HT29, HCT116, and SW48 than in the high expression cells (LS180 and LoVo. This methylation was reversed by treatment with 5-aza-dC, in association with re-expression of PXR and CYP3A4 mRNA, but not VDR mRNA. Therefore, PXR transcription was silenced by promoter methylation in the low expression cells, which most likely led to downregulation of CYP3A4 transactivation. Moreover, a lower level of PXR promoter methylation was observed in colorectal cancer tissues compared with adjacent normal mucosa, suggesting upregulation of the PXR/CYP3A4 mRNAs during carcinogenesis. Conclusions PXR promoter methylation is involved in the regulation of intestinal PXR and CYP3A4 mRNA expression and might be associated with the inter-individual variability

  2. Feed-forward regulation of bile acid detoxification by CYP3A4: studies in humanized transgenic mice.

    Stedman, Catherine; Robertson, Graham; Coulter, Sally; Liddle, Christopher

    2004-03-19

    Bile acids are potentially toxic end products of cholesterol metabolism and their concentrations must be tightly regulated. Homeostasis is maintained by both feed-forward regulation and feedback regulation. We used humanized transgenic mice incorporating 13 kb of the 5' regulatory flanking sequence of CYP3A4 linked to a lacZ reporter gene to explore the in vivo relationship between bile acids and physiological adaptive CYP3A gene regulation in acute cholestasis after bile duct ligation (BDL). Male transgenic mice were subjected to BDL or sham surgery prior to sacrifice on days 3, 6, and 10, and others were injected with intraperitoneal lithocholic acid (LCA) or vehicle alone. BDL resulted in marked hepatic activation of the CYP3A4/lacZ transgene in pericentral hepatocytes, with an 80-fold increase in transgene activation by day 10. Individual bile acids were quantified by liquid chromatography/mass spectrometry. Serum 6beta-hydroxylated bile acids were increased following BDL, confirming the physiological relevance of endogenous Cyp3a induction to bile acid detoxification. Although concentrations of conjugated primary bile acids increased after BDL, there was no increase in LCA, a putative PXR ligand, indicating that this cannot be the only endogenous bile acid mediating this protective response. Moreover, in LCA-treated animals, 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining showed hepatic activation of the CYP3A4 transgene only on the liver capsular surface, and minimal parenchymal induction, despite significant liver injury. This study demonstrates that CYP3A up-regulation is a significant in vivo adaptive response to cholestasis. However, this up-regulation is not dependent on increases in circulating LCA and the role of other bile acids as regulatory molecules requires further exploration. PMID:14681232

  3. Analysis of Mechanism-Based Inhibition of CYP 3A4 by a Series of Fluoroquinolone Antibacterial Agents.

    Watanabe, Akiko; Takakusa, Hideo; Kimura, Takako; Inoue, Shin-Ichi; Kusuhara, Hiroyuki; Ando, Osamu

    2016-10-01

    A series of fluoroquinolone compounds (compounds 1-9), which contain a common quinolone scaffold, inactivated the metabolic activity of CYP3A. The purpose of this study was to identify mechanism-based inhibition (MBI) among these fluoroquinolone compounds by metabolite profiling to elucidate the association of the substructure and MBI potential. Reversibility of MBI after incubation with potassium ferricyanide differed among the test compounds. Representative quasi-irreversible inhibitors form a metabolite-intermediate (MI) complex with the heme of CYP3A4 according to absorption analysis. Metabolite profiling identified the cyclopropane ring-opened metabolites from representative irreversible inhibitors, suggesting irreversible binding of the carbon-centered radical species with CYP3A4. On the other hand, the oxime form of representative quasi-irreversible inhibitors was identified, suggesting generation of a nitroso intermediate that could form the MI complex. Metabolites of compound 10 with a methyl group at the carbon atom at the root of the amine moiety of compound 8 include the oxime form, but compound 10 did not show quasi-irreversible inhibition. The docking study with CYP3A4 suggested that a methyl moiety introduced at the carbon atom at the root of the primary amine disrupts formation of the MI complex between the heme and the nitroso intermediate because of steric hindrance. This study identified substructures of fluoroquinolone compounds associated with the MBI mechanism; introduction of substituted groups inducing steric hindrance with the heme of P450 can prevent formation of an MI complex. Our series of experiments may be broadly applicable to prevention of MBI at the drug discovery stage. PMID:27469000

  4. Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme

    Gnanasekaran, Thiyagarajan; Vavitsas, Konstantinos; Andersen-Ranberg, Johan;

    2015-01-01

    availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase...... enzymes. CONCLUSIONS: It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer...

  5. Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027.

    Van Lanen, Steven G; Lin, Shuangjun; Dorrestein, Pieter C; Kelleher, Neil L; Shen, Ben

    2006-10-01

    C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-beta-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically. PMID:16887797

  6. The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

    Chang, Chung-Te; Bercovich, Natalia; Loh, Belinda; Jonas, Stefanie; Izaurralde, Elisa

    2014-01-01

    The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assem...

  7. Optimization of a novel series of N-phenylindoline-5-sulfonamide-based acyl CoA:monoacylglycerol acyltransferase-2 inhibitors: Mitigation of CYP3A4 time-dependent inhibition and phototoxic liabilities.

    Sato, Kenjiro; Takahagi, Hiroki; Kubo, Osamu; Hidaka, Kousuke; Yoshikawa, Takeshi; Kamaura, Masahiro; Nakakariya, Masanori; Amano, Nobuyuki; Adachi, Ryutaro; Maki, Toshiyuki; Take, Kazumi; Takekawa, Shiro; Kitazaki, Tomoyuki; Maekawa, Tsuyoshi

    2015-08-01

    Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has emerged as a potential peripheral target for the treatment of obesity and metabolic disorders. We previously identified a novel series of N-phenylindoline-5-sulfonamide derivatives exemplified by 2 as potent and orally bioavailable MGAT2 inhibitors. Despite its attractive potency, further assessment revealed that this compound exhibited time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4). To remove the undesirable CYP3A4 TDI activity, structural modification was focused on the 2,4-difluoroaniline moiety on the basis of the assumption that this moiety would be involved in mechanism-based inhibition of CYP3A4 via oxidative metabolism. This led to the finding that the introduction of 4-chloro-2,6-difluoroaniline significantly improved CYP3A4 TDI risk. Further optimization resulted in the discovery of N-(4-chloro-2,6-difluorophenyl)-1-{5-[1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]pyrimidin-2-yl}-7-(2-oxopyrrolidin-1-yl)-2,3-dihydro-1H-indole-5-sulfonamide (27c) with potent MGAT2 inhibitory activity (IC50=7.8 nM) and excellent ADME-Tox profiles including metabolic stability, oral bioavailability, and CYP3A4 TDI. In a mouse oral fat tolerance test, compound 27c effectively and dose-dependently suppressed the elevation of plasma triacylglycerol levels after oral administration at doses of 1 and 3mg/kg. We also discuss mitigation of the phototoxic liability of biaryl derivatives on the basis of the HOMO-LUMO gap hypothesis during the course of optimization efforts. PMID:26100443

  8. Experiment K304: Studies of specific hepatic enzymes and liver constituents involved in the conversion of carbohydrates to lipids in rats exposed to prolonged space flight

    Abraham, S.; Klein, H. P.; Lin, C. Y.; Volkmann, C.; Tigranyan, R. A.; Vetrova, E. G.

    1981-01-01

    The effects of space flight on the activities of 26 enzymes concerned with carbohydrate and lipid metabolism in hepatic tissue taken from male Wistar rats are investigated. These activities were measured in the various hepatic cell compartments, i.e., cytosol, mitochondria and microsomes. In addition, the levels of glycogen, total lipids, phospholipids, triglycerides, cholesterol, cholesterol esters, and the fatty acid composition of the rat livers were also examined and quantified. A similar group of ground-based rats treated in an identical manner served as controls. Both flight and synchronous control rats were sacrificed at three time intervals: R+0, 7-11 hours after recovery; R+6, after 6 days; R+6(S), after 6 days (having undergone 2-5 hour periods of fixed stress in a "backupward" position on days 0, 3, 4, 5 and 6) and R+29, after 29 days post-flight. Although most of the enzyme activities and the amounts of liver constituents studied were unaffected by the period of weightlessness, some significant differences were observed.

  9. Kunstige Enzymer

    Bols, Mikael; Bjerre, Jeannette; Marinescu, Lavinia

    2007-01-01

    Enzymer har en enestående evne til at accelerere kemiske processer. Der forskes målrettet i at optimere enzymer baseret på cyclodextrin.......Enzymer har en enestående evne til at accelerere kemiske processer. Der forskes målrettet i at optimere enzymer baseret på cyclodextrin....

  10. Nitro-Oleic Acid Reduces J774A.1 Macrophage Oxidative Status and Triglyceride Mass: Involvement of Paraoxonase2 and Triglyceride Metabolizing Enzymes.

    Rosenblat, Mira; Rom, Oren; Volkova, Nina; Aviram, Michael

    2016-08-01

    Nitro-fatty acids possess anti-atherogenic properties, but their effects on macrophage oxidative status and lipid metabolism that play important roles in atherosclerosis development are unclear. This study compared the effects of nitro-oleic acid (OLA-NO2) with those of native oleic acid (OLA) on intracellular reactive oxygen species (ROS) generation, anti-oxidants and metabolism of triglycerides and cholesterol in J774A.1 macrophages. Upon incubating the cells with physiological concentrations of OLA-NO2 (0-1 µM) or with equivalent levels of OLA, ROS levels measured by 2, 7-dichlorofluorescein diacetate, decreased dose-dependently, but the anti-oxidative effects of OLA-NO2 were significantly augmented. Copper ion addition increased ROS generation in OLA treated macrophages without affecting OLA-NO2 treated cells. These effects could be attributed to elevated glutathione levels and to increased activity and expression of paraoxonase2 that were observed in OLA-NO2 vs OLA treated cells. Beneficial effects on triglyceride metabolism were noted in OLA-NO2 vs OLA treated macrophages in which cellular triglycerides were reduced due to attenuated biosynthesis and accelerated hydrolysis of triglycerides. Accordingly, OLA-NO2 treated cells demonstrated down-regulation of diacylglycerol acyltransferase1, the key enzyme in triglyceride biosynthesis, and increased expression of hormone-sensitive lipase and adipose triglyceride lipase that regulate triglyceride hydrolysis. Finally, OLA-NO2 vs OLA treatment resulted in modest but significant beneficial effects on macrophage cholesterol metabolism, reducing cholesterol biosynthesis rate and low density lipoprotein influx into the cells, while increasing high density lipoprotein-mediated cholesterol efflux from the macrophages. Collectively, compared with OLA, OLA-NO2 modestly but significantly reduces macrophage oxidative status and cellular triglyceride content via modulation of cellular anti-oxidants and triglyceride

  11. CYP3A4 overexpression enhances apoptosis induced by anticancer agent imidazoacridinone C-1311, but does not change the metabolism of C-1311 in CHO cells

    Pawłowska, Monika; Augustin, Ewa; Mazerska, Zofia

    2013-01-01

    Aim: To examine whether CYP3A4 overexpression influences the metabolism of anticancer agent imidazoacridinone C-1311 in CHO cells and the responses of the cells to C-1311. Methods: Wild type CHO cells (CHO-WT), CHO cells overexpressing cytochrome P450 reductase (CPR) [CHO-HR] and CHO cells coexpressing CPR and CYP3A4 (CHO-HR-3A4) were used. Metabolic transformation of C-1311 and CYP3A4 activity were measured using RP-HPLC. Flow cytometry analyses were used to examine cell cycle, caspase-3 act...

  12. Fluoxetine reduces CES1, CES2, and CYP3A4 expression through decreasing PXR and increasing DEC1 in HepG2 cells.

    Shang, Wei; Liu, Jie; Chen, Ruini; Ning, Rui; Xiong, Jing; Liu, Wei; Mao, Zhao; Hu, Gang; Yang, Jian

    2016-05-01

    1. This study investigated the mechanisms of the decreases of carboxylesterases (CES) and cytochrome P4503A4 (CYP3A4) and the enzymatic activities induced by fluoxetine (FLX) in HepG2 cells. We found that FLX decreased the carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) expression and the hydrolytic activity. 2. FLX decreased the pregnane X receptor (PXR) expression which regulated the target genes such as CYP3A4, whereas increased the differentiated embryonic chondrocyte-expressed gene 1 (DEC1) expression. 3. FLX repressed the PXR at transcriptional level. 4. Overexpression of PXR alone increased the expression of CES1, CES2, and CYP3A4 and attenuated the decreases of CES1, CES2, and CYP3A4 induced by FLX. On the contrary, knockdown of PXR alone decreased the expression of CES1, CES2, and CYP3A4 and almost abolished the decreases of CES1, CES2, and CYP3A4 induced by FLX. 5. Knockdown of DEC1 alone increased the expression of PXR and CYP3A4 and almost abolished the decreases of CES1, CES2, and CYP3A4 induced by FLX. 6. Taken together, the decreases of CES and CYP3A4 expression and enzymatic activities induced by FLX are through decreasing PXR and increasing DEC1 in HepG2 cells. PMID:26340669

  13. Salvianolic acid B modulates the expression of drug-metabolizing enzymes in HepG2 cells

    Qing-LanWang; QuocWu; Yan-Yan Tao; Cheng-Hai Liu; Hani El-Nezami

    2011-01-01

    BACKGROUND: Enzymes involved in drug and xenobiotic metabolism have been considered to exist in two groups: phase I and phase II enzymes. Cytochrome P450 isoenzymes (CYPs) are the most important phase I enzymes in the metabolism of xenobiotics. The products of phase I metabolism are then acted upon by phase II enzymes, including glutathione S-transferases (GSTs). Herbs that inhibit CYPs such as CYP3A4 or that induce GSTs may have the potential to protect against chemical carcinogenesis since the mutagenic effects of carcinogens are often mediated through an excess of CYP-generated reactive intermediates. This study was designed to investigate the effects of salvianolic acid B (Sal B), a pure compound extracted from Radix Salviae Miltiorrhizae, a Chinese herb, on cell proliferation and CYP1A2 and CYP3A4 mRNA expression in the presence or absence of rifampicin, a potent inducer of CYPs and GST protein expression in HepG2 cells. METHODS: HepG2 cells were incubated with different concentrations of Sal B. Cell proliferation was determined by SYTOX-Green nucleic acid staining. CYP3A4 and CYP1A2 mRNA expression was assayed by real-time PCR. GST protein expression was analyzed by Western blotting. RESULTS: Low concentrations of Sal B (0-20 μmol/L) had no significant effects on cell proliferation, while higher concentrations (100-250 μmol/L) significantly inhibited proliferation in a concentration-dependent manner. Tenμmol/L Sal B, but not 1 μmol/L, down-regulated CYP3A4 and CYP1A2 mRNA expression after 24 hours of incubation, whereas both 1 and 10 μmol/L Sal B down-regulated CYP3A4 mRNA expression after 96 hours of incubation; moreover, 1 and 10 μmol/L Sal B inhibited CYP3A4 mRNA expression induced by rifampicin. Both 1 μmol/L and 10 μmol/L Sal B increased GST expression. CONCLUSION: Sal B inhibits CYP3A4 and CYP1A2 mRNA expression and induces GST expression in HepG2 cells.

  14. Comparison of inhibitory duration of grapefruit juice on organic anion-transporting polypeptide and cytochrome P450 3A4.

    Tanaka, Shimako; Uchida, Shinya; Miyakawa, Sachiko; Inui, Naoki; Takeuchi, Kazuhiko; Watanabe, Hiroshi; Namiki, Noriyuki

    2013-01-01

    Recently, a new type of interaction has been reported in which fruit juices diminish oral drug bioavailability through inhibition of organic anion-transporting polypeptide (OATP). In this study, we aimed to clarify the duration of OATP inhibition by grapefruit juice (GFJ), and to compare it with the duration of GFJ-induced inhibition of cytochrome P450 (CYP) 3A4 activity. Seven healthy volunteers were enrolled in this open-label, single-sequence study. They were orally administered celiprolol (100 mg) and midazolam (15 µg/kg) with water on the control day. Three days later, they ingested GFJ (200 mL) 3 times a day for 3 d. On day 1, the same drugs were administered with GFJ. On days 3 and 7, the same drugs were administered with water. Pharmacokinetics of both drugs were evaluated on each trial day. The peak plasma concentration (Cmax) and the area under the plasma concentration-time curve from 0 to 8 h (AUC0-8) of celiprolol significantly decreased on day 1, and the mean ratios of these values and the corresponding control-day values were 0.18 and 0.25, respectively. The Cmax and AUC0-8 returned to the control levels on days 3 and 7. In contrast, AUC0-8 of midazolam were higher on days 1 and 3 than on the control day (mean ratio, 2.12 and 1.47, respectively). The AUC0-8 returned to the control level on day 7. In conclusion, results of this study indicated that the OATP inhibition caused by GFJ dissipated faster than GFJ-mediated alterations in CYP3A4 activity, which were sustained for at least 48 h. PMID:24292052

  15. Possible Involvement of Anti-Oxidant Enzymes in the Cross-Tolerance of the Germination/Growth of Wheat Seeds to Salinity and Heat Stress

    Yan-Bao LEI; Song-Quan SONG; Jia-Rui FU

    2005-01-01

    The germination/growth of wheat (Triticun aestivum L. cv. Zimai 1) seeds and changes in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), as well as in the content of thiobarbituric acid-reactive substances (TBARS), in response to salt and heat stress, as well as cross-stress, were investigated in the present study. With increasing temperature and decreasing water potential caused by NaC1 solution, the germination percentage of seeds and the fresh weight of seedlings decreased markedly, SOD activity increased, activities of APX and CAT decreased distinctly, and the TBARS content increased gradually. Seeds pretreated at 33 ℃ for different times displayed increased tolerance to subsequent salt stress, enhanced SOD, APX, and CAT activities, and decreased TBARS content. Seeds pretreated at -0.8 MPa NaC1 for different times displayed increased tolerance to subsequent heat stress and marked increases in SOD, APX, and CAT activities, which were associated with decreased TBARS content. It is considered that the common component in the cross-tolerance of the germination and growth of wheat seeds to salinity and heat stress is the anti-oxidant enzyme system.

  16. An RNA-binding complex involved in ribosome biogenesis contains a protein with homology to tRNA CCA-adding enzyme.

    Jinzhong Lin

    2013-10-01

    Full Text Available A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA-protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.

  17. Inhibition of Vibrio harveyi bioluminescence by cerulenin: In vivo evidence for covalent modification of the reductase enzyme involved in aldehyde synthesis

    Byers, D.M. (Dalhousie Univ., Halifax (Nova Scotia)); Meighen, E.A. (McGill Univ., Montreal, Quebec (Canada))

    1989-07-01

    Bacterial bioluminescence is very sensitive to cerulenin, a fungal antibiotic which is known to inhibit fatty acid synthesis. When Vibrio harveyi cells pretreated with cerulenin were incubated with ({sup 3}H)myristic acid in vivo, acylation of the 57-kilodalton reductase subunit of the luminescence-specific fatty acid reductase complex was specifically inhibited. Light emission of wild-type V. harveyi was 20-fold less sensitive to cerulenin at low concentrations (10{mu}g/ml) than that of the dark mutant strain M17, which requires exogenous myristic acid for luminescence because of a defective transferase subunit. The sensitivity of myristic acid-stimulated luminescence in the mutant strain M17 exceeded that of phospholipid synthesis from ({sup 14}C)acetate, whereas uptake and incorporation of exogenous ({sup 14}C)myristic acid into phospholipids was increased by cerulenin. The reductase subunit could be labeled by incubating M17 cells with ({sup 3}H)tetrahydrocerulenin; this labeling was prevented by preincubation with either unlabeled cerulenin or myristic acid. Labeling of the reductase subunit with ({sup 3}H)tetrahydrocerulenin was also noted in an aldehyde-stimulated mutant (A16) but not in wild-type cells or in another aldehyde-stimulated mutant (M42) in which ({sup 3}H)myristoyl turnover at the reductase subunit was found to be defective. These results indicate that (i) cerulenin specifically and covalently inhibits the reductase component of aldehyde synthesis, (ii) this enzyme is partially protected from cerulenin inhibition in the wild-type strain in vivo, and (iii) two dark mutants which exhibit similar luminescence phenotypes (mutants A16 and M42) are blocked at different stages of fatty acid reduction.

  18. Inhibition of Vibrio harveyi bioluminescence by cerulenin: In vivo evidence for covalent modification of the reductase enzyme involved in aldehyde synthesis

    Bacterial bioluminescence is very sensitive to cerulenin, a fungal antibiotic which is known to inhibit fatty acid synthesis. When Vibrio harveyi cells pretreated with cerulenin were incubated with [3H]myristic acid in vivo, acylation of the 57-kilodalton reductase subunit of the luminescence-specific fatty acid reductase complex was specifically inhibited. Light emission of wild-type V. harveyi was 20-fold less sensitive to cerulenin at low concentrations (10μg/ml) than that of the dark mutant strain M17, which requires exogenous myristic acid for luminescence because of a defective transferase subunit. The sensitivity of myristic acid-stimulated luminescence in the mutant strain M17 exceeded that of phospholipid synthesis from [14C]acetate, whereas uptake and incorporation of exogenous [14C]myristic acid into phospholipids was increased by cerulenin. The reductase subunit could be labeled by incubating M17 cells with [3H]tetrahydrocerulenin; this labeling was prevented by preincubation with either unlabeled cerulenin or myristic acid. Labeling of the reductase subunit with [3H]tetrahydrocerulenin was also noted in an aldehyde-stimulated mutant (A16) but not in wild-type cells or in another aldehyde-stimulated mutant (M42) in which [3H]myristoyl turnover at the reductase subunit was found to be defective. These results indicate that (i) cerulenin specifically and covalently inhibits the reductase component of aldehyde synthesis, (ii) this enzyme is partially protected from cerulenin inhibition in the wild-type strain in vivo, and (iii) two dark mutants which exhibit similar luminescence phenotypes (mutants A16 and M42) are blocked at different stages of fatty acid reduction

  19. SUMO and SUMO-Conjugating Enzyme E2 UBC9 Are Involved in White Spot Syndrome Virus Infection in Fenneropenaeus chinensis.

    Xiaoqian Tang

    Full Text Available In previous work, small ubiquitin-like modifier (SUMO in hemocytes of Chinese shrimp Fenneropenaeus chinensis was found to be up-regulated post-white spot syndrome virus (WSSV infection using proteomic approach. However, the role of SUMO in viral infection is still unclear. In the present work, full length cDNAs of SUMO (FcSUMO and SUMO-conjugating enzyme E2 UBC9 (FcUBC9 were cloned from F. chinensis using rapid amplification of cDNA ends approach. The open reading frame (ORF of FcSUMO encoded a 93 amino acids peptide with the predicted molecular weight (M.W of 10.55 kDa, and the UBC9 ORF encoded a 160 amino acids peptide with the predicted M.W of 18.35 kDa. By quantitative real-time RT-PCR, higher mRNA transcription levels of FcSUMO and FcUBC9 were detected in hemocytes and ovary of F. chinensis, and the two genes were significantly up-regulated post WSSV infection. Subsequently, the recombinant proteins of FcSUMO and FcUBC9 were expressed in Escherichia coli BL21 (DE3, and employed as immunogens for the production of polyclonal antibody (PAb. Indirect immunofluorescence assay revealed that the FcSUMO and UBC9 proteins were mainly located in the hemocytes nuclei. By western blotting, a 13.5 kDa protein and a 18.7 kDa protein in hemocytes were recognized by the PAb against SUMO or UBC9 respectively. Furthermore, gene silencing of FcSUMO and FcUBC9 were performed using RNA interference, and the results showed that the number of WSSV copies and the viral gene expressions were inhibited by knockdown of either SUMO or UBC9, and the mortalities of shrimp were also reduced. These results indicated that FcSUMO and FcUBC9 played important roles in WSSV infection.

  20. MicroRNA-30c-1-3p is a silencer of the pregnane X receptor by targeting the 3'-untranslated region and alters the expression of its target gene cytochrome P450 3A4.

    Vachirayonstien, Thaveechai; Yan, Bingfang

    2016-09-01

    The pregnane X receptor (PXR) is a master regulator of genes involved in drug elimination. Recently, activation of PXR has also been linked to the development of many disease conditions such as metabolic disorders and malignancies. MicroRNAs (miRs) emerge as important molecular species involved in these conditions. This study was undertaken to test a large number of miRs for their ability to regulate PXR expression. As many as 58 miRs were tested and miR-30c-1-3p was identified to suppress PXR expression. The suppression was achieved by targeting the 3'-untranslated region, 438 nucleotides from the stop codon. The suppression was detected in multiple cell lines from different organ origins. In addition, miR-30c-1-3p altered basal and induced expression of cytochrome P450 3A4 (CYP3A4), a prototypical target gene of PXR. The alteration varied depending on the time and amounts of miR-30c-1-3p. CYP3A4 is responsible for the metabolism of more than 50% medicines. The interconnection between miR-30c-1-3p and PXR signifies a role of miRs in drug-drug interactions and chemosensitivity. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. PMID:27085140

  1. Prednisone has no effect on the pharmacokinetics of CYP3A4 metabolized drugs - midazolam and odanacatib.

    Marcantonio, Eugene E; Ballard, Jeanine; Gibson, Christopher R; Kassahun, Kelem; Palamanda, Jairam; Tang, Cuyue; Evers, Raymond; Liu, Chengcheng; Zajic, Stefan; Mahon, Chantal; Mostoller, Kate; Hreniuk, David; Mehta, Anish; Morris, Denise; Wagner, John A; Stoch, S Aubrey

    2014-11-01

    We evaluated the effect of prednisone on midazolam and odanacatib pharmacokinetics. In this open-label, 2-period crossover study in healthy male subjects, midazolam 2 mg was administered (Day -1) followed by odanacatib 50 mg (Day 1) during Part 1. In Period 2, prednisone 10 mg once daily (qd) was administered on Days 1-28; odanacatib was co-administered on Day 14 and midazolam 2 mg was co-administered on Days 1 and 28. Subjects were administered midazolam 2 mg on Days 42 and 56. Safety and tolerability were assessed throughout the study. A physiologically-based pharmacokinetic (PBPK) model was also built. There were 15 subjects enrolled; mean age was 31 years. The odanacatib AUC(0- ∞) GMR (90% CI) [odanacatib + prednisone (Day 14, Period 2)/odanacatib alone (Day 1, Period 1] was 1.06 (0.96, 1.17). AUC(0-∞) GMR (90%CI) [midazolam + prednisone (Day 28, Period 2)/midazolam alone (Day -1, Period 1] was 1.08 (0.93,1.26). There were no serious AEs or AEs leading to discontinuation. PBPK modeling showed that prednisone does not cause significant effects on the exposure of sensitive CYP3A4 substrates in vivo at therapeutic doses. Co-administration of prednisone 10 mg qd had no effect on pharmacokinetics of either odanacatib 10 mg or midazolam 2 mg. PMID:24895078

  2. Content of CYP3A4 inhibitors, naringin, naringenin and bergapten in grapefruit and grapefruit juice products.

    Ho, P C; Saville, D J; Coville, P F; Wanwimolruk, S

    2000-04-01

    The flavonoids, naringin and naringenin and the furanocoumarin, bergapten (5-methoxypsoralen), were detected in some fresh grapefruit and commercial grapefruit juices but were not detected in other fruit juices tested (orange; orange with apple base; dark grape; orange and mango with apple base; orange, peach, passion fruit juice). The contents of these three grapefruit constituents in commercial juice and fresh grapefruit varied from brand to brand and also from lot to lot. Juice was prepared from the fresh fruit via different methods (by hand, squeezer or blender). The naringin content, after hand-squeeze, ranged from 115 to 384 mg/l. With hand-squeeze juice production, bergapten was not detected (less than 0.5 mg/l) in two varieties of grapefruit, and naringenin was usually not in detectable levels (less than 2 mg/l) in three varieties. All three constituents were present in New Zealand grapefruit preparations (including juice by hand-squeeze) and different lots showed variation in content (1.5-, 2.3- and 4.7-fold for naringin, naringenin and bergapten, respectively). Differences in the concentrations of these three constituents, which have potential for drug interaction, may contribute to the variability in pharmacokinetics of CYP3A4 drugs and some contradictory results of drug interaction studies with grapefruit juice. PMID:10812937

  3. Enzymes involved in apoptosis of medical protozoan%原虫凋亡中相关酶类研究进展

    杨秋林(综述); 许丽芳(审校)

    2015-01-01

    近年鉴定到Metacaspase、组织蛋白酶B、组织蛋白酶D、核酸酶( Endo G、Tatd 、Fen-1)等分子参与了原虫的凋亡,但不清楚这些分子在凋亡信号途径中的位置及相互关系。实验结果显示,Metacaspase可能具有调节原虫凋亡与细胞周期等功能,但是Metacaspase与Caspase的活化方式及作用底物不同,提示原虫存在与多细胞动物不同的凋亡途径;在疟原虫及利什曼原虫中发现其线粒体及溶酶体参与了其凋亡,提示原虫可能具有类似哺乳动物的溶酶体-线粒体凋亡途径;在利什曼原虫和锥虫中发现存在通过核酸酶而不依赖Caspase的凋亡途径。阐明原虫的凋亡机制有助于通过设计新药物诱导原虫凋亡来达到治疗疾病的目的。%Recently, a number of molecules which are involved in the apoptosis in protozoa have been discovered, for ex-ample:metacaspase, cathepsin B and D, nuclease( Endo G, Tatd, Fen-1 et al) .So far the locations of these molecules in the signal pathway of protozoan apoptosis and the relationship associated to these molecules in the signal pathway have not been identified.The results of experiments showed that the activation mode is different between metacaspase and caspase. The substrates are also different for metacaspase and caspase.The results suggested that the apoptosis mechanism is different between protozoan and metazoan.Metacaspase has multifunction, for example: control of apoptosis, cell cycle and clear-ance of insoluble protein aggregates.The results of experiment showed that mitochondrial and lysosomal body are involved in the apoptosis in protozoan, possibly, there are mitochondrial-lysosomal body pathway and caspase-independent pathway which control the apoptosis in protozoan.Uncovering clearly the mechanism of apoptosis in protozoan is benefit for design of new drugs in prevention and treatment of diseases caused by the protozoa.

  4. The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa-zona pellucida interaction.

    Moreno, R D; Sepúlveda, M S; de Ioannes, A; Barros, C

    1998-02-01

    Mammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellucida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains of the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation. PMID:9652074

  5. Thiolate exchange in [TmR]ZnSR' complexes and relevance to the mechanisms of thiolate alkylation reactions involving zinc enzymes and proteins.

    Melnick, Jonathan G; Zhu, Guang; Buccella, Daniela; Parkin, Gerard

    2006-05-01

    The zinc and cadmium thiolate complexes [TmBut]MSCH2C(O)N(H)Ph (M = Zn, Cd) may be obtained via treatment of the respective methyl complex [TmBut]MMe with PhN(H)C(O)CH2SH. The molecular structure of [TmBut]ZnSCH2C(O)N(H)Ph has been determined by X-ray diffraction, thereby demonstrating the presence of an intramolecular N-H S hydrogen bond between the amide N-H group and thiolate sulfur atom. [TmBut]ZnSCH2C(O)N(H)Ph mimics the function of the Ada DNA repair protein by undergoing alkylation with MeI to give [TmBut]ZnI and MeSCH2C(O)N(H)Ph. A series of crossover experiments and 1H NMR magnetization transfer studies establish that thiolate exchange between [TmR]ZnSR' derivatives is facile in this system, an observation that supports the previous suggestion that the alkylation of [TmPh]ZnSCH2C(O)N(H)Ph by MeI may proceed via a sequence that involves dissociation of [PhN(H)C(O)CH2S]-. PMID:16516971

  6. NRC integrated program for the resolution of Unresolved Safety Issues A-3, A-4 and A-5 regarding steam generator tube integrity: Final report

    This report presents the results of the NRC integrated program for the resolution of Unresolved Safety Issues (USIs) A-3, A-4, and A-5 regarding steam generator tube integrity. A generic risk assessment is provided and indicates that risk from steam generator tube rupture (SGTR) events is not a significant contributor to total risk at a given site, nor to the total risk to which the general public is routinely exposed. This finding is considered to be indicative of the effectiveness of licensee programs and regulatory requirements for ensuring steam generator tube integrity in accordance with 10 CFR 50, Appendices A and B. This report also identifies a number of staff-recommended actions that the staff finds can further improve the effectiveness of licensee programs in ensuring the integrity of steam generator tubes and in mitigating the consequences of an SGTR. As part of the integrated program, the staff issued Generic Letter 85-02 encouraging licensees of pressurized water reactors (PWRs) to upgrade their programs, as necessary, to meet the intent of the staff-recommended actions; however, such actions do not constitute NRC requirements. In addition, this report describes a number of ongoing staff actions and studies involving steam generator issues which are being pursued to provide added assurance that risk from SGTR events will continue to be small. 146 refs., 5 figs., 11 tabs

  7. Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells: An insight into the efflux-metabolism alliance.

    Hua, Fang; Shi, Mei-jun; Zhu, Xiao-lu; Li, Meng; Wang, Hong-xu; Yu, Xiao-ming; Li, Yan; Zhu, Chuan-jiang

    2015-11-01

    The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide (CLA) enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients (Papp) of (-) and (+)CLA were higher in the absorptive direction than those in the secretory direction with efflux ratios (ER) of 0.709±0.411 and 0.867±0.250 (×10(-6)cm/s), respectively. Their bidirectional transports were significantly reduced by 75.6-87.5% after treatment with verapamil (a P-glycoprotein inhibitor) that increased the rate of metabolism by CYP3A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of (-) and (+)CLA with ERs being 2.934±1.432 and 1.877±0.148(×10(-6)cm/s) respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enantiomers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following (-) or (+)-isomer treatment alone. Furthermore, the uptake of (-)CLA was more than that of (+)CLA in the transfected cells. Incubation with ketoconazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexamethasone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharmacology of (-)CLA as a candidate drug for treatment of Alzheimer's disease. PMID:26301745

  8. Evaluation of 4β-Hydroxycholesterol as a Clinical Biomarker of CYP3A4 Drug Interactions Using a Bayesian Mechanism–Based Pharmacometric Model

    Leil, T A; Kasichayanula, S; Boulton, D W; LaCreta, F

    2014-01-01

    A Bayesian mechanism–based pharmacokinetic/pharmacodynamic model of cytochrome P450 3A4 (CYP3A4) activity was developed based on a clinical study of the effects of ketoconazole and rifampin on midazolam exposure and plasma 4β-hydroxycholesterol (4βHC) concentrations. Simulations from the model demonstrated that the dynamic range of 4βHC as a biomarker of CYP3A4 induction or inhibition was narrower than that of midazolam; an inhibitor that increases midazolam area under the curve by 20-fold ma...

  9. How Does the Reductase Help To Regulate the Catalytic Cycle of Cytochrome P450 3A4 Using the Conserved Water Channel?

    Fishelovitch, Dan; Shaik, Sason; Wolfson, Haim J.; Nussinov, Ruth

    2010-01-01

    Water molecules play a major role in the P450 catalytic cycle. Here, we locate the preferred water pathways and their gating mechanisms for the human cytochrome P450 3A4 (CYP3A4) and elucidate the role of the cytochrome P450 reductase (CPR) in turning on and activating these water channels. We perform explicit solvent molecular dynamic simulations of CYP3A4, unbound and bound to two substrates, and with and without the flavin mononucleotide (FMN)-binding domain of CPR. We observe in/out passa...

  10. Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

    Flueck, Christa E.; Mullis, Primus E. [Pediatric Endocrinology, Diabetology and Metabolism, Department of Clinical Research, University of Bern, Tiefenaustrasse 120c, CH 3004 Bern (Switzerland); Pandey, Amit V., E-mail: amit@pandeylab.org [Pediatric Endocrinology, Diabetology and Metabolism, Department of Clinical Research, University of Bern, Tiefenaustrasse 120c, CH 3004 Bern (Switzerland)

    2010-10-08

    Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

  11. Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

    Research highlights: → Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). → Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. → We are reporting that mutations in POR may reduce CYP3A4 activity. → POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. → Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

  12. N-Terminal Region of GbIspH1, Ginkgo biloba IspH Type 1, May Be Involved in the pH-Dependent Regulation of Enzyme Activity

    Bok-Kyu Shin

    2015-01-01

    Full Text Available GbIspH1, IspH type 1 in Ginkgo biloba chloroplast, is the Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP and dimethylallyl diphosphate (DMAPP at the final step of methylerythritol phosphate pathway in chloroplast. Compared to the bacterial IspH, plant IspH, including GbIspH1, has an additional polypeptide chain at the N-terminus. Here, biochemical function of the N-terminal region of GbIspH1 was investigated with the N-terminal truncated GbIspH1 (GbIspH1-truncated. Both wild type GbIspH1 (GbIspH1-full and GbIspH1-truncated were catalytically active and produced IPP and DMAPP in a ratio of 15 : 1. Kinetic parameters of KM (17.3 ± 1.9 and 14.9 ± 2.3 µM and kcat (369 ± 10 and 347 ± 12 min−1 at pH 8.0 were obtained for GbIspH1-full and GbIspH1-truncated, respectively. Interestingly, GbIspH1-full and GbIspH1-truncated showed significantly different pH-dependent activities, and the maximum enzyme activities were obtained at pH 8.0 and 7.5, respectively. However, catalytic activation energies (Ea of GbIspH1-full and GbIspH1-truncated were almost the same with 36.5 ± 1.6 and 35.0 ± 1.9 kJ/mol, respectively. It was suggested that the N-terminal region of GbIspH1 is involved in the pH-dependent regulation of enzyme activity during photosynthesis.

  13. Racial Differences in CYP3A4 Genotype and Survival Among Men Treated on Radiation Therapy Oncology Group (RTOG) 9202: A Phase III Randomized Trial

    Purpose: Inherited genotypes may explain the inferior outcomes of African American (AA) men with prostate cancer. To understand how variation in CYP3A4 correlated with outcomes, a retrospective examination of the CYP3A4*1B genotype was performed on men treated with Radiation Therapy Oncology Group (RTOG) 92-02. Methods and Materials: From 1,514 cases, we evaluated 56 (28.4%) of 197 AA and 54 (4.3%) of 1,274 European American (EA) patients. All patients received goserelin and flutamide for 2 months before and during RT (STAD-RT) ± 24 months of goserelin (long-term androgen deprivation plus radiation [LTAD-RT]). Events studied included overall survival and biochemical progression using American Society for Therapeutic Radiology and Oncology consensus guidelines. Results: There were no differences in outcome in patients in with or without CYP3A4 data. There was an association between race and CYP3A4 polymorphisms with 75% of EAs having the Wild Type compared to only 25% of AA men (p <0.0001). There was no association between CYP3A4 classification or race and survival or progression. Conclusions: The samples analyzed support previously reported observations about the distribution of CYP3A4*1B genotype by race, but race was not associated with poorer outcome. However, patient numbers were limited, and selection bias cannot be completely ruled out

  14. Enzymes involved in a degradation of explosives

    Vaněk, Tomáš; Nepovím, Aleš

    Athens : Agricultural University of Athens, 2000, s. 88-91. [COST Action 837. Phytoremediation 2000 State of the Art in Europe. Scientific Workshop /1./. Hersonissos (GR), 06.04.2000-08.04.2000] Institutional research plan: CEZ:AV0Z4055905 Subject RIV: CE - Biochemistry

  15. Oxidative enzymes involved in organic xenobiotic metabolism

    Harvey, P. J.; Werck-Reichhart, D.; Chaudhry, M. Q.; Coyle, C.; Vaněk, Tomáš

    Athens : Agricultural University of Athens, 2000, s. 65-68. [COST Action 837. Phytoremediation 2000 State of the Art in Europe. Scientific Workshop /1./. Hersonissos (GR), 06.04.2000-08.04.2000] Institutional research plan: CEZ:AV0Z4055905 Subject RIV: CE - Biochemistry

  16. The independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and the metabolism of atorvastatin.

    Liu, Ju-E; Ren, Bin; Tang, Lan; Tang, Qian-Jie; Liu, Xiao-Ying; Li, Xin; Bai, Xue; Zhong, Wan-Ping; Meng, Jin-Xiu; Lin, Hao-Ming; Wu, Hong; Chen, Ji-Yan; Zhong, Shi-Long

    2016-01-01

    To evaluate the independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and atorvastatin metabolism, the relationships among three levels of factors, namely (1) clinical characteristics, CYP3A4/5 genotypes, and miRNAs, (2) CYP3A4 and CYP3A5 mRNAs, and (3) CYP3A activity, as well as their individual impacts on atorvastatin metabolism, were assessed in 55 human liver tissues. MiR-27b, miR-206, and CYP3A4 mRNA respectively accounted for 20.0%, 5.8%, and 9.5% of the interindividual variations in CYP3A activity. MiR-142 was an independent contributor to the expressions of CYP3A4 mRNA (partial R(2) = 0.12, P = 0.002) and CYP3A5 mRNA (partial R(2) = 0.09, P = 0.005) but not CYP3A activity or atorvastatin metabolism. CYP3A activity was a unique independent predictor of variability of atorvastatin metabolism, explaining the majority of the variance in reduction of atorvastatin (60.0%) and formation of ortho-hydroxy atorvastatin (78.8%) and para-hydroxy atorvastatin (83.9%). MiR-27b and miR-206 were found to repress CYP3A4 gene expression and CYP3A activity by directly binding to CYP3A4 3'-UTR, while miR-142 was found to indirectly repress CYP3A activity. Our study indicates that miRNAs play significant roles in bridging the gap between epigenetic effects and missing heritability in CYP3A functionality. PMID:27211076

  17. The Effect of microRNAs in the Regulation of Human CYP3A4: a Systematic Study using a Mathematical Model

    Wei, Zhiyun; Jiang, Songshan; Zhang, Yiting; Wang, Xiaofei; Peng, Xueling; Meng, Chunjie; Liu, Yichen; Wang, Honglian; Guo, Luo; Qin, Shengying; He, Lin; Shao, Fengmin; Zhang, Lirong; Xing, Qinghe

    2014-03-01

    CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations.

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

    Nik Nur Syazana Bt Nik Mohamed Kamal

    2013-01-01

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

  19. Protein Phosphatase 2Cβl Regulates Human Pregnane X Receptor-Mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells

    Pondugula, Satyanarayana R.; Tong, Alexander A.; Wu, Jing; Cui, Jimmy; Chen, Taosheng

    2010-01-01

    The human pregnane X receptor (hPXR) regulates the expression of CYP3A4, which plays a vital role in hepatic drug metabolism and has considerably reduced expression levels in proliferating hepatocytes. We have recently shown that cyclin-dependent kinase 2 (CDK2) negatively regulates hPXR-mediated CYP3A4 gene expression. CDK2 can be dephosphorylated and inactivated by protein phosphatase type 2C beta isoform long (PP2Cβl), a unique phosphatase that was originally cloned from human liver. In th...

  20. TRANSFECTED MDCK CELL LINE WITH ENHANCED EXPRESSION OF CYP3A4 AND P-GLYCOPROTEIN AS A MODEL TO STUDY THEIR ROLE IN DRUG TRANSPORT AND METABOLISM

    Kwatra, Deep; Budda, Balasubramanyam; Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Ashim K. Mitra

    2012-01-01

    The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drugs of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Simila...

  1. A Comprehensive in vitro and in silico Analysis of Antibiotics that Activate PXR and Induce CYP3A4 in Liver and Intestine

    Yasuda, Kazuto; Ranade, Aarati; Venkataramanan, Raman; Strom, Stephen; Chupka, Jonathan; Ekins, Sean; Schuetz, Erin; Bachmann, Kenneth

    2008-01-01

    We have investigated several in silico and in vitro methods in order to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate PXR and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative PCR, immunobl...

  2. Different effects of proton pump inhibitors and famotidine on the clopidogrel metabolic activation by recombinant CYP2B6, CYP2C19 and CYP3A4.

    Ohbuchi, Masato; Noguchi, Kiyoshi; Kawamura, Akio; Usui, Takashi

    2012-07-01

    Inhibitory potential of proton pump inhibitors (PPIs) and famotidine, an H(2) receptor antagonist, on the metabolic activation of clopidogrel was evaluated using recombinant CYP2B6, CYP2C19 and CYP3A4. Formation of the active metabolite from an intermediate metabolite, 2-oxo-clopidogrel, was investigated by liquid chromatography-tandem mass spectrometry and three peaks corresponding to the pharmacologically active metabolite and its stereoisomers were detected. Omeprazole potently inhibited clopidogrel activation by CYP2C19 with an IC(50) of 12.8 μmol/L and more weakly inhibited that by CYP2B6 and CYP3A4. IC(50) of omeprazole for CYP2C19 and CYP3A4 was decreased about two- and three-fold, respectively, by 30-min preincubation with NADPH. Lansoprazole, esomeprazole, pantoprazole, rabeprazole and rabeprazole thioether, a major metabolite, also inhibited metabolic activation by CYP2C19, with an IC(50) of 4.3, 8.9, 48.3, 36.2 and 30.5 μmol/L, respectively. In contrast, famotidine showed no more than 20% inhibition of clopidogrel activation by CYP2B6, CYP2C19 and CYP3A4 at up to 100 μmol/L and had no time-dependent CYP2C19 and CYP3A4 inhibition. These results provide direct evidence that PPIs inhibit clopidogrel metabolic activation and suggest that CYP2C19 inhibition is the main cause of drug-drug interaction between clopidogrel and omeprazole. Famotidine is considered as a safe anti-acid agent for patients taking clopidogrel. PMID:22313038

  3. Enzyme immunoassay

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from...

  4. Food Enzymes

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  5. Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

    Xiao-mei ZHUANG; Jing-ting DENG; Hua LI; Wei-li KONG; Jin-xiu RUAN; Lan XIE

    2011-01-01

    Aim:To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK),a novel anti-HIV agent,by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs).Methods:CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2,2B6,2C8,2C9,2C19,2D6,3A4,and 3A5.LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites.In the experiments with recombinant cytochrome P450 enzymes,specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism.Results:CMDCK was rapidly and extensively metabolized by HLMs.Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL.min-1·kg-1,which was comparable to the mean human hepatic blood flow rate (20.7 mL·min-1·kg-1).The major metabolic pathway of CMDCK was oxidation,and a total of 14 metabolites were detected.CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism.Conclusion:CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites.CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.

  6. Catabolism of methyl ter-butyl ether (MTBE): characterization of the enzymes of Mycobacterium austroafricanum IFP 2012 involved in MTBE degradation; Catabolisme du methyl tert-butyl ether (MTBE): caracterisation des enzymes impliquees dans la degradation du MTBE chez Mycobacterium austroafricanum IFP 2012

    Lopes Ferreira, N.

    2005-11-15

    Methyl tert-butyl ether (MTBE) is added to gasoline to meet the octane index requirement. its solubility in water and its poor biodegradability made the use of MTBE a great environmental concern, particularly regarding aquifers. We previously isolated M austroafricanum IFP 2012 able to use MTBE as a sole source of carbon and energy and the MTBE pathway was partially characterized. In the present study, which aimed at isolating the genes involved in MTBE biodegradation in order to use them for estimation of MTBE biodegradation capacities in contaminated environment, we isolated a new M. austroafricanum strain, IFP 2015. A new degradation intermediate, the 2-methyl 1,2-propane-diol (2-M1,2-PD), the product of tert-butanol (TBA) oxidation, was identified. We also determined the enzymes induced during growth of M. austroafricanum IFP 2012 on MTBF. Then, using the tools of protein analysis and of molecular biology, we isolated and cloned the mpd genes cluster in the plasmid pCL4D. Heterologous expression of the recombinant plasmid in M smegmatis tmc2 155, showed the involvement of an 2-M1,2-PD dehydrogenase (MpdB) and a hydroxy-iso-butyr-aldehyde dehydrogenase (MpdC), encoded by mpdB and mpdC, respectively. Both enzymes were responsible for the conversion of 2-M 1,2-PD to hydroxy-isobutyric acid (HIBA). A further survey of different M austroafricanum strains, including IFP 2012, IFP 2015 and JOBS (ex-M vaccae) showed the link between the ability to grow on C{sub 2} to C{sub 16} n-alkanes and the MTBE and TBA degradation capacities. The alkB gene was partially sequenced in all these strains. Expression of alkB was demonstrated in M. austroafricanum IFP 2012 after growth on propane, hexane, hexadecane and TBA. Finally, we identified 2-propanol as the intermediate of HIBA degradation. The gene encoding the 2-propanol:p-N,N'-dimethyl-4-nitroso-aniline (NDMA) oxidoreductase was detected M austroafricanum IFP 2012. (author)

  7. Trial of the correlation between cytochrome oxidase CYP3A4 with the susceptibility of paclitaxel-based regimen for advanced gastric cancer

    Jianwei Yang; Yan Meng; Ying Su; Zeng Chen; Wei Gao; Jinyuan Lin; Jing Jia; Huamei Lin

    2014-01-01

    Objective: The aim of the study was to investigate the relationship between susceptibility of paclitaxel-based regimen and gene polymorphisms of cytochrome oxidase CYP3A4 for advanced gastric cancer.Methods: Peripheral venous blood sample of 53 advanced gastric cancer patients were enroled to test the mutation of CYP3A4 gene by denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. The relation between the eficacy of paclitaxel-based regimen and CYP3A4 gene polymorphisms was further analyzed.Results: DHPLC indicated that among the 53 patients, 21 cases showed biomodal type (mutation) and 32 cases were of unimodal type (wild-type). Sequencing results showed that the deletion mutation was found at the 27th basic group of C in exon 10 of CYP3A4 gene. The response rate (RR) and disease control rate (DCR) of wild-type group were 40.6% and 84.4%, while in mutation group they were 33.3% and 85.7%, respective-ly, with no significances between the two groups (P > 0.05). Of al 53 cases, the median progression-free survival (PFS) was 6.5 months (95% CI: 3.576-9.424 months), and the median overal survival (OS) was 11.0 months (95% CI: 6.955-15.045 months). The median PFS and OS in wild-type group had no diferences compared with those in mutation group (7.0 months vs. 7.0 months,P > 0.05; 10.0 months vs. 14.0 months,P > 0.05). Between wild-type and mutation groups, the median PFS of patients applied with oxaliplatin containing regimen and the median OS in patients applied with/without oxaliplatin had no significant diferences (P > 0.05), while the median PFS in patients received non-oxaliplatin regime had statistical diferences (P = 0.024). The median PFS and OS in patients receiving 3-drug or 2-drug regimes had no correlation with CYP3A4 gene polymorphisms. The adverse efects in the two groups were mild, mainly in grades 1-2. The common adverse efects were anorexia, nausea/vomiting and leucopenia.Conclusion: Deletion mutation was located in the 27th

  8. Association between cytochrome CYP17A1, CYP3A4, and CYP3A43 polymorphisms and prostate cancer risk and aggressiveness in a Korean study population

    Jun Hyun Han

    2015-04-01

    Full Text Available In this study, we evaluated genetic variants of the androgen metabolism genes CYP17A1, CYP3A4, and CYP3A43 to determine whether they play a role in the development of prostate cancer (PCa in Korean men. The study population included 240 pathologically diagnosed cases of PCa and 223 age-matched controls. Among the 789 single-nucleotide polymorphism (SNP database variants detected, 129 were reported in two Asian groups (Han Chinese and Japanese in the HapMap database. Only 21 polymorphisms of CYP17A1, CYP3A4, and CYP3A43 were selected based on linkage disequilibrium in Asians (r2 = 1, locations (SNPs in exons were preferred, and amino acid changes and were assessed. In addition, we performed haplotype analysis for the 21 SNPs in CYP17A1, CYP3A4, and CYP3A43 genes. To determine the association between genotype and haplotype distributions of patients and controls, logistic analyses were carried out, controlling for age. Twelve sequence variants and five major haplotypes were identified in CYP17A1. Five sequence variants and two major haplotypes were identified in CYP3A4. Four sequence variants and four major haplotypes were observed in CYP3A43. CYP17A1 haplotype-2 (Ht-2 (odds ratio [OR], 1.51; 95% confidence interval [CI], 1.04-2.18 was associated with PCa susceptibility. CYP3A4 Ht-2 (OR: 1.87; 95% CI: 1.02-3.43 was associated with PCa metastatic potential according to tumor stage. rs17115149 (OR: 1.96; 95% CI: 1.04-3.68 and CYP17A1 Ht-4 (OR: 2.01; 95% CI: 1.07-4.11 showed a significant association with histologic aggressiveness according to Gleason score. Genetic variants of CYP17A1 and CYP3A4 may play a role in the development of PCa in Korean men.

  9. Expression and Clinical Significance of P-gp and CYP3A4 in Diffuse Large B-cell Lymphoma%P-gp和CYP3A4在弥漫性大B细胞淋巴瘤中的表达及临床意义

    张发良; 王子安; 陶言言; 毕国斌

    2012-01-01

    Objective:To study the expression of P - glycoprotein ( P-gp) and cytochromeP4503A4 (CYP3A4) in diffuse large B - cell lymphoma ( DLBCL) : to explore the role of the two proteins in multidrug resistance and the clinical significance in DLBCL. Methods: 60 cases of DLBCL paraffin wax samples were selected. Immunohistochemistry was used to examine the expression of P-gp, CYP3A4 respectively. The relationship between P-gp, CYP3A4 and clinicopathological parameters, chemosensitivity and prognosis of DLBCL cases were analyzed. Results: 1. The positive expression rate of P-gp in DLBCL tissues was 33.3% and the positive expression rate of CYP3A4 was 58.3%. 2. Significant positive correlation was found between the expressions of P-gp and CYP3A4 in DLBCL tissues(rs =0.392,P<0.05). 3. The chemotherapy ORR was significantly lower in P-gp +/CYP3A4 + team than that in P-gp -/CYP3A4 - team ( P <0.05) , but the refractory rate was significantly higher in P-gp +/CYP3A4 + team than that in P-gp -/CYP3A4 - team ( P <0.05). 4. The overall survival of P-gp - team, CYP3A4 - team were longer than that of P-gp + team, CYP3A4 + team. The diversity between the two teams had statistical significance ( P < 0.05 ). Conclusions: 1. There is a higher expression of P-gp, CYP3A4 in DLBCL tissues, and the two proteins may be useful markers of prognosis in DLBCL 2. P-gp and CYP3A4 are important indicators to estimate Chemo - sensitivity in DLBCL, and there is a synergistic effect between P-gp and CYP3A4 on multidrug resistance in DLBCL. So detecting aggregated expression of the two proteins may have an important sense to select effective drug and set individualized chemotherapy regimens.%目的:研究P-糖蛋白(P-gp)和细胞色素P450 3A4( CYP3A4)在弥漫性大B细胞淋巴瘤( DLBCL)中的表达情况,并探讨二者在DLBCL多药耐药中的作用.方法:收集60例DLBCL石蜡标本,采用免疫组织化学方法检测其P-gp、CYP3A4的表达情况,并分析二者的相关性及其与DLBCL国

  10. Photoreactivating enzymes

    Photoreactivating enzymes (PRE) also called photolyases (EC 4.1.99.3) catalyze the light 300 to 600 nm)-dependent monomerization of cyclobutyl pyrimidine dimers, formed between adjacent pyrimidines on the same DNA strand, upon exposure to ultraviolet (uv) irradiation (220 to 320 nm). Although much is known about the substrate and product of these unusual enzymes, their identification required the development and synthesis of such fields as photochemistry, biochemistry, and microbiology. Photoreactivation was first known as a biological recovery phenomenon: cells exposed to visible light following uv irradiation showed higher survival than those kept in the dark. Early investigators examined the photoreactivability of an enormous range of cellular damage in both prokaryotes and eukaryotes. This review article discusses the purification and properties of PRE, the kinetics of photoreactivation and the biological role of this repair process

  11. Effects of salvianolic acid B and tanshinone IIA on the pharmacokinetics of losartan in rats by regulating the activities and expression of CYP3A4 and CYP2C9.

    Wang, Rong; Zhang, Hai; Wang, Yujie; Yu, Xiaoyan; Yuan, Yongfang

    2016-03-01

    Losartan (LST) is a common chemical drug used to treat high blood pressure and reduce the risk of stroke in certain people with heart disease. Danshen, prepared from the dried root and rhizome of Salvia miltiorrhiza Bunge, has been widely used for prevention and treatment of various cardiovascular and cerebrovascular diseases. There are more than 35 formulations containing Danshen indexed in the 2010 Chinese Pharmacopoeia, which are often combined with LST to treat cardiovascular and cerebrovascular diseases in the clinic. The effects of the two major components of Danshen, salvianolic acid B (SA-B) and tanshinone IIA (Tan IIA), on the pharmacokinetics of losartan and its metabolite, EXP3174, in rats were investigated by liquid chromatography coupled with mass spectrometry (LC-MS). Male Sprague-Dawley rats were randomly assigned to 3 groups: LST, LST+SA-B and LST+Tan IIA, and the main pharmacokinetic parameters were estimated after oral administration of LST, LST+SA-B and LST+Tan IIA. It was found that there are significant differences in the pharmacokinetic parameters among the three groups: Cmax, t1/2, AUC, AUMC in the LST+SA-B group was smaller than those in group LST, while larger in group LST+Tan IIA. Further, the effects of SA-B and Tan IIA on the metabolism of losartan was also investigated using rat liver microsomes in vitro. The results indicated that SA-B can induce the metabolism of LST, while Tan IIA can inhibit the metabolism of LST in rat liver microsomes in vitro by regulating activities of CYP450 enzymes. In addition, the effect of SA-B and Tan IIA on CYP3A4 and CYP2C9 expression was studied in Chang liver cells by western-blotting and Real-time PCR. It was concluded that the two components of Danshen, SA-B and Tan IIA have different influences on the metabolism of LST: SA-B can obviously speed up the metabolism of LST by inducing CYP3A4/CYP2C9 activities and expression, however, Tan IIA can slow down the metabolism of LST by inhibiting CYP3A4/CYP2C

  12. Engineering enzymes

    Dutton, P. Leslie; Moser, Christopher C.

    2011-01-01

    Fundamental research into bioinorganic catalysis of the kind presented at this Faraday Discussion has the potential to turn inspiration drawn from impressive natural energy and chemical transformations into artificial catalyst constructions useful to mankind. Creating bio-inspired artificial constructions requires a level of understanding well beyond simple description of structures and mechanisms of natural enzymes. To be useful, such description must be augmented by a practical sense of str...

  13. Effect of ketoconazole-mediated CYP3A4 inhibition on clinical pharmacokinetics of panobinostat (LBH589), an orally active histone deacetylase inhibitor

    Hamberg, Paul; Woo, Margaret M.; Chen, Lin-Chi; Verweij, Jaap; Porro, M.G.; Zhao, Ling; Li, Weili; van der Biessen, Diane; Sharma, Hari; Hengelage, Thomas; Jonge, Maja

    2011-01-01

    Abstract Purpose Panobinostat is partly metabolized by CYP3A4 in vitro. This study evaluated the effect of a potent CYP3A inhibitor, ketoconazole, on the pharmacokinetics and safety of panobinostat. Methods Patients received a single panobinostat oral dose on day 1, followed by 4 days wash-out period. On days 5?9, ketoconazole was administered. On day 8, a single panobinostat dose was co-administered with ...

  14. Two surfaces of cytochrome b5 with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries.

    Peng, Hwei-Ming; Auchus, Richard J

    2014-01-01

    Conserved human cytochrome b5 (b5) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b5 mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b5 double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23-42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b5, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b5 but the poorest activity with b5 mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl]>50mM. Cross-linking of CYP3A4 and b5 decreased in the order wild-type>E48G/E49G>D58G/D65G and varied with phospholipid. We conclude that two b5 acidic surfaces, primarily the domain including residues D58-D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b5 mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4. PMID:24256945

  15. Activation of interfacial enzymes at membrane surfaces

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi; Hansen, Per Lyngs; Jakobsen, Ask F.; Bernchou Jensen, Uffe; Jensen, Morten Ø.; Jørgensen, Kent; Kaasgaard, Thomas; Leidy, Chad; Simonsen, Adam Cohen; Peters, Günther H.J.; Weiss, Matthias

    2006-01-01

    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s...

  16. Immobilization of Enzymes in Polymer Supports.

    Conlon, Hugh D.; Walt, David R.

    1986-01-01

    Two experiments in which an enzyme is immobilized onto a polymeric support are described. The experiments (which also demonstrate two different polymer preparations) involve: (1) entrapping an enzyme in an acrylamide polymer; and (2) reacting the amino groups on the enzyme's (esterase) lysine residues with an activated polymer. (JN)

  17. Lignolytic Enzymes Production from Selected Mushrooms

    H.M. Shantaveera Swamy

    2015-06-01

    Full Text Available In this paper, ligninase enzymes produced by selected mushrooms have been reported. We collected mushrooms from Western Ghats, most of them were edible food. Thirty samples isolated were tested using a plate assay through direct agar plate assay by using ABTS, decolourisation containing the fifteen isolates were able to decolourise the dye, indicating a lignin-degrading ability. Spectrophotometric enzyme assays from all selected isolates were carried out to examine the production of Ligninolytic enzymes (Laccase, lignin peroxidase and manganese peroxidase. Ten selected isolates produced all three kinds of enzymes tested. Lignolytic enzymes are groups of enzymes these are actively involved in bioremediation.

  18. Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays.

    Guengerich, F Peter; Wheeler, James B; Chun, Young-Jin; Kim, Donghak; Shimada, Tsutomu; Aryal, Pramod; Oda, Yoshimitsu; Gillam, Elizabeth M J

    2002-12-27

    Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. PMID:12505322

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

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

    2011-08-01

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

  20. Effect of ketoconazole-mediated CYP3A4 inhibition on clinical pharmacokinetics of panobinostat (LBH589), an orally active histone deacetylase inhibitor

    Hamberg, Paul; Woo, Margaret M.; Chen, Lin-Chi; Verweij, Jaap; Porro, M.G.; Zhao, Ling; Li, Weili; van de Biessen, D; Sharma, Hari; Hengelage, Thomas; Jonge, Maja

    2011-01-01

    textabstractPurpose: Panobinostat is partly metabolized by CYP3A4 in vitro. This study evaluated the effect of a potent CYP3A inhibitor, ketoconazole, on the pharmacokinetics and safety of panobinostat. Methods: Patients received a single panobinostat oral dose on day 1, followed by 4 days wash-out period. On days 5-9, ketoconazole was administered. On day 8, a single panobinostat dose was co-administered with ketoconazole. Panobinostat was administered as single agent three times a week on d...

  1. 26种中药提取物对CYP3A4和CYP2D6代谢的抑制作用

    Iwata; H; 郑晓燕(摘译); 阴赪宏(校)

    2005-01-01

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

  2. Positron emitter labeled enzyme inhibitors

    This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

  3. Process for preparing multilayer enzyme coating on a fiber

    Kim, Jungbae; Kwak, Ja Hun; Grate, Jay W.

    2009-11-03

    A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  4. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p Solanum paniculatum (-44%, p Solanum paniculatum.

  5. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  6. Spectroscopic studies and molecular docking on the interaction of organotin antitumor compound bis[2,4-difluoro-N-(hydroxy-⟨κ⟩O)benzamidato-⟨κ⟩O]diphenyltin(IV) with human cytochrome P450 3A4 protease

    Wei, Ying; Niu, Lin; Liu, Xinxin; Zhou, Hongyan; Dong, Hongzhou; Kong, Depeng; Li, Yunlan; Li, Qingshan

    2016-06-01

    A novel organotin DFDPT was synthesized and characterized by elemental analysis, IR, 1H, 13C, 119Sn, NMR techniques,etc. In order to investigate profoundly the relationship between DFDPT with human CYP3A4 proteaset and anticancer molecular mechanism of DFDPT, the intercalative mode of binding of DFDPT with CYP3A4 under physiological conditions were comprehensively evaluated using steady state, synchronous, three-dimensional fluorescence spectroscopy,circular dichroism and molecular docking. Fluorescence emission data showed that CYP3A4 fluorescence affected by DFDPT was a static quenching procedure, which implied that DFDPT-CYP3A4 complex had been formed. Apparent binding constants Kb of CYP3A4 with compound at 298 and 310 K were 2.51 × 107 and 3.09 × 105, respectively. The binding sites number n was 1.64 and 1.22, respectively. The thermodynamic parameters ΔH and ΔS of the DFDPT-CYP3A4 complex were negative, which indicated that their interaction was driven mainly by hydrogen bonding and van der Waals force. The binding of DFDPT-CYP3A4 was spontaneous process in which ΔG was negative. The synchronous results showed DFDPT induced conformational changes of CYP3A4 protein. Three-dimensional fluorescence and circular dichroism spectra results also revealed conformation of CYP3A4 protein had been possible changed in the presence of DFDPT. Molecular docking was used to study the interaction orientation between DFDPT and CYP3A4 protease. The results indicated that DFDPT interacted with a panel of amino acids in the active sites of CYP3A4 protein mainly through formation of hydrogen bond. Furthermore, the predicted binding mode of DFDPT into CYP3A4 appeared to adopt an orientation with interactions among Arg105, Ser119 and Thr309.

  7. Spectroscopic studies and molecular docking on the interaction of organotin antitumor compound bis[2,4-difluoro-N-(hydroxy-⟨κ⟩O)benzamidato-⟨κ⟩O]diphenyltin(IV) with human cytochrome P450 3A4 protease.

    Wei, Ying; Niu, Lin; Liu, Xinxin; Zhou, Hongyan; Dong, Hongzhou; Kong, Depeng; Li, Yunlan; Li, Qingshan

    2016-06-15

    A novel organotin DFDPT was synthesized and characterized by elemental analysis, IR, (1)H, (13)C, (119)Sn, NMR techniques,etc. In order to investigate profoundly the relationship between DFDPT with human CYP3A4 proteaset and anticancer molecular mechanism of DFDPT, the intercalative mode of binding of DFDPT with CYP3A4 under physiological conditions were comprehensively evaluated using steady state, synchronous, three-dimensional fluorescence spectroscopy,circular dichroism and molecular docking. Fluorescence emission data showed that CYP3A4 fluorescence affected by DFDPT was a static quenching procedure, which implied that DFDPT-CYP3A4 complex had been formed. Apparent binding constants Kb of CYP3A4 with compound at 298 and 310K were 2.51×10(7) and 3.09×10(5), respectively. The binding sites number n was 1.64 and 1.22, respectively. The thermodynamic parameters ΔH and ΔS of the DFDPT-CYP3A4 complex were negative, which indicated that their interaction was driven mainly by hydrogen bonding and van der Waals force. The binding of DFDPT-CYP3A4 was spontaneous process in which ΔG was negative. The synchronous results showed DFDPT induced conformational changes of CYP3A4 protein. Three-dimensional fluorescence and circular dichroism spectra results also revealed conformation of CYP3A4 protein had been possible changed in the presence of DFDPT. Molecular docking was used to study the interaction orientation between DFDPT and CYP3A4 protease. The results indicated that DFDPT interacted with a panel of amino acids in the active sites of CYP3A4 protein mainly through formation of hydrogen bond. Furthermore, the predicted binding mode of DFDPT into CYP3A4 appeared to adopt an orientation with interactions among Arg105, Ser119 and Thr309. PMID:27049867

  8. 定量5-羟奥美拉唑和奥美拉唑砜以探测中国人肝微粒体中CYP2C19和CYP3A4的活性%Probing CYP2C19 and CYP3A4 activities in Chinese liver microsomes by quantification of 5-hydroxyomeprazole and omeprazole suiphone

    舒焱; 王连生; 肖卫民; 王伟; 黄松林; 周宏灏

    2000-01-01

    AIM: To develop an analytical method for simultaneous quantification of 5-hydroxyomeprazole (5-OH-OP) and omeprazole suffone (OPS), and explore whether omeprazole (OP) is an appropriate phenotypic probe for CYP2C19 and CYP3A4 in Chinese liver microsomes.METHODS: OP metabolism in vitro was conducted in Chinese liver microsomes, and the major metabolites 5-OH-OP and OPS were determined using high pressure liquid chromatography (HPLC). Monoclonal antibodies anti-CYP2C8/9/19 and anti-CYP3A4 were employed to conduct inhibition experiments. The protein contents of CYP2C19 and CYP3A4 were quantified using Western blot analysis and densitometric scanning. RESULTS:5-OH-OP and OPS gave a baseline resolution in the HPLC analysis. The detection limits for both compounds were 0.01 nmol and the recovery (98 % -102 %) had good precision with relative standard deviation of 87 % ). At a substrate concentration of 2 μmol/L OP, good correlations were found between OP 5-hydroxylation and S-mephenytoin 4'-hydroxylation activities ( r = 0.72, P 87%);在底物浓度为2 μmol/L OP时,中国人肝微粒体中OP的5-羟化与美芬妥英的4'-羟化活性之间(r=0.72,P<0.01)、OP的5-羟化活性与CYP2C19含量之间(r=0.82,P<0.01)以及OP的硫代氧化活性与CYP3A4含量之间(r=0.78,P<0.01)均有很好的相关性.结论:中国人肝微粒体中OP的代谢主要由CYP2C19和CYP3A4介导;采用本研究建立的HPLC方法,在适当的底物浓度下,OP能用于体外探测中国人肝微粒体中CYP2C19及CYP3A4的活性.

  9. In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import

    Peterson, Nathan A; Tavis K Anderson; Wu, Xiao-Jun; Yoshino, Timothy P.

    2013-01-01

    Background Carbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose...

  10. 淫羊藿总黄酮对大鼠肝微粒体CYP1A2、CYP3A4和CYP2E1活性的影响%Effect of total flavonoids of epimedium on liver microsomal CYP1A2, CYP3A4 and CYP2E1 activities in rats

    胡道德; 姚慧娟; 顾磊; 王松坡; 刘皋林

    2008-01-01

    To assess the potential effect of total flavonoids of epimedium (TFE) on cytochrome P450 and activity of its main isoforms in rat liver microsomes. TFE (300 mg/kg) was administered once daily to male Sprague-Dowley rats by gavage for fifteen days. The total cytochrome P450 content and its main isoforms CYP1A2, CYP3A4 and CYP2E1 activities in rat liver microsomes were detected. The activity of CYP1A2 was measured by fluorometry and the activities of CYP3A4 and CYP2E1 were determined by measuring the amount of methanal and p-aminophenol formed using UV/Vis spectrophotometer, respec- tively. Administration of TFE significantly increased the total CYP450 content and activities of CYP 1A2, CYP3A4 and CYP2E1 in rat liver microsomes, compared with the control group. Partieularly, the activities of CYP1A2 and CYP2E1 were enhanced significantly (P<0.01). TFE induced the increase in total CYP450 content and its main isoforms CYP1A2, CYP3A4 and CYP2EI activities in rat liver microsomes.%评估淫羊藿总黄酮对大鼠肝细胞色素P450及其主要亚型活性的潜在影响.淫羊藿总黄酮以300 mg/kg/d的剂量对SD大鼠进行连续灌胃处理15天,测定肝微粒体中CYP450含量与CYP1A2、CYP3A4和CYP2E1亚型活性,观察淫羊藿总黄酮的效应.CYP1A2的活性用荧光比色法进行测定,CYP3A4和CYP2E1的活性用紫外可见分光光度法测定.淫羊藿总黄酮处理后的大鼠肝脏CYP450含量及CYP1A2、CYP3A4和LICYP2E1亚型活性均明显增高,其中CYP1A2和CYP2E1活性升高显著(P<0.01).淫羊藿总黄酮对大鼠肝脏CYP450及主要亚型CYP1A2、CYP3A4和CYP2E1活性均有诱导效应.

  11. Enzyme detection by microfluidics

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted by that...... enzyme...

  12. Quorum quenching enzymes.

    Fetzner, Susanne

    2015-05-10

    Bacteria use cell-to-cell communication systems based on chemical signal molecules to coordinate their behavior within the population. These quorum sensing systems are potential targets for antivirulence therapies, because many bacterial pathogens control the expression of virulence factors via quorum sensing networks. Since biofilm maturation is also usually influenced by quorum sensing, quenching these systems may contribute to combat biofouling. One possibility to interfere with quorum sensing is signal inactivation by enzymatic degradation or modification. Such quorum quenching enzymes are wide-spread in the bacterial world and have also been found in eukaryotes. Lactonases and acylases that hydrolyze N-acyl homoserine lactone (AHL) signaling molecules have been investigated most intensively, however, different oxidoreductases active toward AHLs or 2-alkyl-4(1H)-quinolone signals as well as other signal-converting enzymes have been described. Several approaches have been assessed which aim at alleviating virulence, or biofilm formation, by reducing the signal concentration in the bacterial environment. These involve the application or stimulation of signal-degrading bacteria as biocontrol agents in the protection of crop plants against soft-rot disease, the use of signal-degrading bacteria as probiotics in aquaculture, and the immobilization or entrapment of quorum quenching enzymes or bacteria to control biofouling in membrane bioreactors. While most approaches to use quorum quenching as antivirulence strategy are still in the research phase, the growing number of organisms and enzymes known to interfere with quorum sensing opens up new perspectives for the development of innovative antibacterial strategies. PMID:25220028

  13. Distribution of cytochrome P450 2C, 2E1, 3A4, and 3A5 in human colon mucosa

    Parlesak, Alexandr

    2005-01-01

    BACKGROUND: Despite the fact that the alimentary tract is part of the body's first line of defense against orally ingested xenobiotica, little is known about the distribution and expression of cytochrome P450 (CYP) enzymes in human colon. Therefore, expression and protein levels of four represent...

  14. It takes two to tango: two TatA paralogues and two redox enzyme-specific chaperones are involved in the localization of twin-arginine translocase substrates in Campylobacter jejuni.

    Liu, Y.W.; Hitchcock, A.; Salmon, R.C.; Kelly, D J

    2014-01-01

    The food-borne zoonotic pathogen Campylobacter jejuni has complex electron transport chains required for growth in the host, many of which contain cofactored periplasmic enzymes localized by the twin-arginine translocase (TAT). We report here the identification of two paralogues of the TatA translocase component in C. jejuni strain NCTC 11168, encoded by cj1176c (tatA1) and cj0786 (tatA2). Deletion mutants constructed in either or both of the tatA1 and tatA2 genes displayed distinct growth an...

  15. Dynamically simulating the interaction of midazolam and the CYP3A4 inhibitor itraconazole using individual coupled whole-body physiologically-based pharmacokinetic (WB-PBPK models

    Jang In-Jin

    2007-03-01

    Full Text Available Abstract Background Drug-drug interactions resulting from the inhibition of an enzymatic process can have serious implications for clinical drug therapy. Quantification of the drugs internal exposure increase upon administration with an inhibitor requires understanding to avoid the drug reaching toxic thresholds. In this study, we aim to predict the effect of the CYP3A4 inhibitors, itraconazole (ITZ and its primary metabolite, hydroxyitraconazole (OH-ITZ on the pharmacokinetics of the anesthetic, midazolam (MDZ and its metabolites, 1' hydroxymidazolam (1OH-MDZ and 1' hydroxymidazolam glucuronide (1OH-MDZ-Glu using mechanistic whole body physiologically-based pharmacokinetic simulation models. The model is build on MDZ, 1OH-MDZ and 1OH-MDZ-Glu plasma concentration time data experimentally determined in 19 CYP3A5 genotyped adult male individuals, who received MDZ intravenously in a basal state. The model is then used to predict MDZ, 1OH-MDZ and 1OH-MDZ-Glu concentrations in an CYP3A-inhibited state following ITZ administration. Results For the basal state model, three linked WB-PBPK models (MDZ, 1OH-MDZ, 1OH-MDZ-Glu for each individual were elimination optimized that resulted in MDZ and metabolite plasma concentration time curves that matched individual observed clinical data. In vivo Km and Vmax optimized values for MDZ hydroxylation were similar to literature based in vitro measures. With the addition of the ITZ/OH-ITZ model to each individual coupled MDZ + metabolite model, the plasma concentration time curves were predicted to greatly increase the exposure of MDZ as well as to both increase exposure and significantly alter the plasma concentration time curves of the MDZ metabolites in comparison to the basal state curves. As compared to the observed clinical data, the inhibited state curves were generally well described although the simulated concentrations tended to exceed the experimental data between approximately 6 to 12 hours following

  16. The enzymes associated with denitrification

    Hochstein, L. I.; Tomlinson, G. A.

    1988-01-01

    The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes. This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases.

  17. Herbal medicine Yin Zhi Huang induces CYP3A4-mediated sulfoxidation and CYP2C19-dependent hydroxylation of omeprazole

    Lan FAN; Hong-hao ZHOU; Guo WANG; Lian-sheng WANG; Yao CHEN; Wei ZHANG; Yuan-fei HUANG; Rui-xue HUANG; Dong-li HU; Dan WANG

    2007-01-01

    Aim: To explore the potential interactions between Yin Zhi Huang (YZH) and omeprazole, a substrate of CYP3A4 and CYP2C19. Methods: Eighteen healthy volunteers, including 6 CYP2C19* 1/*1, 6 CYP2C19*1/*2 or *3 and 6 CYP2C19*2/ *2 were enrolled in a 2-phase, randomized, crossover clinical trial. In each phase,the volunteers received either placebo or 10 mL YZH oral liquid, 3 times daily for 14 d. Then all the patients took a 20 mg omeprazole capsule orally. Blood samples were collected up to 12 h after omeprazole administration. Plasma concentrations of omeprazole and its metabolites were quantified by HPLC with UV detection.Results: After 14 d of treatment of YZH, plasma omeprazole significantly decreased and those of omeprazole sulfone and 5-hydroxyomeprazole signifi-cantly increased. The ratios of the area under the plasma concentration-time curves from time 0 to infinity (AUC(0-∞) of omeprazole to 5-hydroxyomprazole and those of omeprazole to omeprazole sulfone decreased by 64.80%±12.51% (P=0.001 ) and 63.31%±18.45 % (P=0.004) in CYP2C 19* 1/* 1,57.98%±14. 80% (P=0.002)and 54.87%±18.42% (P=0.003) in CYP2C19*1/*2 or *3, and 37.74%±16.07% (P=0.004) and 45.16%± 15.54% (P=0.003) in CYP2C19*2/*2, respectively. The decrease of the AUC(0-∞) ratio of omeprazole to 5-hydroxyomprazole in CYP2C19*1/*1 and CYP2C19*1/*2 or *3 was greater than those in CYP2C19*2/*2 (P=0.047 and P=0.009). Conclusion: YZH induces both CYP3A4-catalyzed sulfoxidation and CYP2C19-dependent hydroxylation of omeprazole leading to decreases in plasma omeprazole concentrations.

  18. Effects of pectin pentaoligosaccharide from Hawthorn ( Crataegus pinnatifida Bunge. var. Major) on the activity and mRNA levels of enzymes involved in fatty acid oxidation in the liver of mice fed a high-fat diet.

    Li, Tuo-Ping; Zhu, Ru-Gang; Dong, Yin-Ping; Liu, Yong-Hui; Li, Su-Hong; Chen, Gang

    2013-08-01

    The regulatory effects of haw pectin pentaoligosaccharide (HPPS) on fatty acid oxidation-related enzyme activities and mRNA levels were investigated in the liver of high fat diet induced hyperlipidemic mice. Results showed that HPPS (150 mg/kg for 10 weeks) significantly suppresses weight gain (32.3 ± 0.26 and 21.1 ± 0.14 g for high-fat diet and HPPS groups, respectively), decreases serum triacylglycerol levels (1.64 ± 0.09 and 0.91 ± 0.02 mmol/L, respectively), and increases lipid excretion in feces (55.7 ± 0.38 and 106.4 ± 0.57 mg/g for total lipid, respectively), compared to high-fat diet as control. HPPS significantly increased the hepatic fatty acid oxidation-related enzyme activities of acyl-CoA oxidase, carnitine palmitoyltransferase I, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase by 53.8, 74.2, 47.1, and 24.2%, respectively. Meanwhile, the corresponding mRNAs were up-regulated by 89.6, 85.8, 82.9, and 30.9%, respectively. Moreover, HPPS was able to up-regulate the gene and protein expressions of peroxisome proliferator-activated receptor α. Results suggest that continuous HPPS ingestion may be used as dietary therapy to prevent obesity and cardiovascular diseases. PMID:23855516

  19. Hemolytic Effects of Melittin Involve Activity Change of Two Types of Enzymes on RBC Membrane%蜂毒肽的溶血作用与红细胞膜上两种酶活性变化的关系

    赵亚华; 李日清; 张微; 钟杨生; 梁祖承; 林健荣

    2008-01-01

    从蜂毒肽作用于红细胞膜上的Na+-K+-ATPase和葡萄糖-6-磷酸脱氢酶(G-6-PD)活性变化的角度,利用分光光度法测定酶活性,研究蜂毒肽与红细胞及膜作用过程中可能的靶点,讨论了蜂毒肽溶血过程与RBC膜上2种酶活性的变化.结果发现,蜂毒肽抑制RBC膜上酶活性的主要模式为附着/插入质膜与游离态并存模式,附着/插入质膜中的作用大于游离态的作用.Na+-K+-ATPase的K+结合位点是蜂毒肽的1个作用靶点.蜂毒肽插膜过程与其对此酶的作用随时间延长同步发生.蜂毒肽通过作用于葡萄糖-6-磷酸和NADP使G-6-PD的催化受到缓慢抑制,蜂毒肽形成四聚体的程度与酶活性密切相关.EDTA抑制蜂毒肽聚集,干扰蜂毒肽作用于G-6-P,蜂毒肽作用于底物G-6-P及辅酶NADP的生化机理相似,蜂毒肽抑制作用与G-6-PD的结构无关.%The effects of melittin on the activities of Na+-K+-ATPase and glucose-6-phosphate dehydrogenase (G-6-PD) which are on the membrane of red blood cell (RBC) are chosen as the index of this study. The possible target sites of these effects through enzyme activity determination by spectrophotometry are investigated, and the hemolytic process and the activity change of these two types of enzymes on the RBC membrane are discussed. The results show that the main mode of melittin inhibition to the activity of enzymes on the RBC membrane is the coexistence of adhesion/insertion form and free-state form, and the effect of the former is more stronger than the latter. The K+ binding site of Na+-K+-ATPase is one of the target sites of melittin. The membrane-insertion process of melittin synchronizes with the action of melittin on this enzyme. Melittin slowly inhibits the catalysis of G-6-PD through the action on G-6-P and NADP, and the extent in which melittin forms tetramers isclosely related to the enzyme activity. EDTA inhibits the aggregation of melittin, and interferes with its action on G-6-P. The

  20. Enzyme immobilization: an update

    Homaei, Ahmad Abolpour; Sariri, Reyhaneh; Vianello, Fabio; Stevanato, Roberto

    2013-01-01

    Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immo-bilized enzyme systems allows an easy recovery of both enzymes and products, multiple re-use of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This paper is a review of the recent literatures on enzyme immobilization by various techniques, the need...

  1. Haplotypes in the APOA1-C3-A4-A5 gene cluster affect plasma lipids in both humans and baboons

    Wang, Qian-fei; Liu, Xin; O' Connell, Jeff; Peng, Ze; Krauss, Ronald M.; Rainwater, David L.; VandeBerg, John L.; Rubin, Edward M.; Cheng, Jan-Fang; Pennacchio, Len A.

    2003-09-15

    Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human QTL genes in a highly controlled non-human primate model.

  2. Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.

    Ploux, O; Breyne, O; Carillon, S; Marquet, A

    1999-01-01

    8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using

  3. Chemical and functional characterization of seed, pulp and skin powder from chilto (Solanum betaceum), an Argentine native fruit. Phenolic fractions affect key enzymes involved in metabolic syndrome and oxidative stress.

    Orqueda, María Eugenia; Rivas, Marisa; Zampini, Iris Catiana; Alberto, María Rosa; Torres, Sebastian; Cuello, Soledad; Sayago, Jorge; Thomas-Valdes, Samanta; Jiménez-Aspee, Felipe; Schmeda-Hirschmann, Guillermo; Isla, María Inés

    2017-02-01

    The aim of this work was to assess the nutritional and functional components of powder obtained by lyophilization of whole fruits, seeds, pulp and skin from chilto (Solanum betaceum Cav) cultivated in the ecoregion of Yungas, Argentina. The powders have low carbohydrate and sodium content and are a source of vitamin C, carotenoid, phenolics, potassium and fiber. The HPLC-ESI-MS/MS analysis of the fractions enriched in phenolics allowed the identification of 12 caffeic acid derivatives and related phenolics, 10 rosmarinic acid derivatives and 7 flavonoids. The polyphenols enriched extracts before and after simulated gastroduodenal digestion inhibited enzymes associated with metabolic syndrome, including α-glucosidase, amylase and lipase and exhibited antioxidant activity by different mechanisms. None of the analyzed fruit powders showed acute toxicity or genotoxicity. The powders from the three parts of S. betaceum fruit may be a potential functional food and the polyphenol enriched extract of seed and skin may have nutraceutical properties. PMID:27596394

  4. Starch-related Enzymes during Potato Tuber Dormancy and Sprouting

    Sergeeva, L.I.; Claassens, M.M.J.; Jamar, D.C.L.; Plas, van der L.H.W.; Vreugdenhil, D.

    2012-01-01

    Activities of enzymes presumably involved in starch biosynthesis (ADP-glucose pyrophosphorylase, AGPase) and/or breakdown (starch phosphorylase, STP; amylases) were determined during potato (Solanum tuberosum L.) tuber dormancy and sprouting. Overall activities of all these enzymes decreased during

  5. Epoxidation of the methamphetamine pyrolysis product, trans-phenylpropene, to trans-phenylpropylene oxide by CYP enzymes and stereoselective glutathione adduct formation

    Pyrolytic products of smoked methamphetamine hydrochloride are well established. Among the various degradation products formed, trans-phenylpropene (trans-β-methylstyrene) is structurally similar to styrene analogues known to be bioactivated by CYP enzymes. In human liver microsomes, trans-phenylpropene was converted to the epoxide trans-phenylpropylene oxide (trans-2-methyl-3-phenyloxirane) and cinnamyl alcohol. Incubation of trans-phenylpropene with microsomes in the presence of enzyme-specific P450 enzyme inhibitors indicated the involvement of CYP2E1, CYP1A2, and CYP3A4 enzymes. Both (R,R)-phenylpropylene oxide and (S,S)-phenylpropylene oxide were formed in human liver microsomal preparations. Enantiomers of trans-phenylpropylene oxide were stereoselectively and regioselectively conjugated in a Phase II drug metabolism reaction catalyzed by human liver cytosolic enzymes consisting of conjugation with glutathione. The structure of the phenylpropylene oxide-glutathione adduct is consistent with nucleophilic ring-opening by attack at the benzylic carbon. Exposure of cultured C6 glial cells to (S,S)-phenylpropylene oxide produced a cytotoxic response in a concentration-dependent manner based on cell degeneration and death

  6. EXTRACCIÓN DE ENZIMAS PÉCTICAS DEL EPICARPIO DE LULO (Solanum quitoense Lam INVOLUCRADAS EN EL PROCESO DE ABLANDAMIENTO Exytraction of Pectic Enzymes from of Lulo(Solanum quitoense Lam Involved in Softening

    JEIMMY MARCELA RODRÍGUEZ NIETO

    2011-08-01

    Full Text Available Durante el periodo de poscosecha el principal problema de deterioro del lulo (Solanum quitoense Lam es el ablandamiento que es generado principalmente por actividad de enzimas pécticas que atacan la red estructural de la pared celular. Esta investigación se basó en la búsqueda de las mejores condiciones de extracción y medida de actividad de las enzimas pectinesterasa, poligalacturonasa y pectato liasa; herramientas necesarias para estudiar posteriormente el rol de estas enzimas en el deterioro por ablandamiento sufrido por el fruto debido a diversos cambios metabólicos. Se encontró que las dos primeras enzimas pueden ser extraídas simultáneamente con buffer fosfatos 20 mM pH 7,0 + NaCl 0,06 M y 60 min de extracción, relación 1:2 (material vegetal: buffer de extracción, a su vez, pectato liasa se extrajo con buffer fosfatos 20 mM pH 7,0 + cisteína 20 mM y 30 min de extracción, relación 1:3. Para la cuantificación de la actividad pectinesterasa es necesario incubar 15 min a 42 °C 2.500 µL de extracto enzimático crudo (EE en buffer fosfatos 20 mM pH 7,0 + NaCl 0,15 M y 1,6% de pectina cítrica como sustrato, con valores de Km aparente de 3,78% de PC y Vmax 17,95 µmolH+/min*mg prot. Para la cuantificación de la actividad poligalacturonasa es necesario incubar 15 min a 37 °C 30 µL (EE en buffer acetatos 200 mM pH 4,5 + NaCl 0,25 M y 1,0% de APG como sustrato, con valores de Km aparente 0,141% de APG y Vmax 28,46 nKat/s*mg prot. Para la cuantificación de la actividad pectato liasa es necesario incubar 2 min a 17 °C 100 µL (EE en buffer TRIS:HCl 50 Mm pH 8,5 + CaCl2 4 mM y 0,1% de APG como sustrato, con valores de Km aparente 0,0865% de APG y Vmax 82,75 µg/s*mg prot.The main problem of post-harvest deterioration of lulo (Solanum quitoense Lam is the softening is the main problem of post-harvest deteriorarion of Lulo, that is generated mainly by the activity of pectic enzymes, which attack the structural network of the cell

  7. Efeitos da incorporação de peneiras moleculares 3A, 4A, 5A e 13X em membranas compósitas de quitosana/poli(vinil álcool Effect of molecular sieves 3A, 4A, 5A and 13X incorporation on the chitosan/poly(vinyl alcohol composites membranes

    Denice Schulz Vicentini

    2010-01-01

    Full Text Available The composite membranes prepared via incorporation of 12.5% of molecular sieves 3A, 4A, 5A and 13X into chitosan/poly(vinyl alcohol (1:1. The composite membranes were immersed in the cross-linker sulfuric acid in order to acquire high proton conductivity for applications in electrolytes in PEMCF and DMF. The influence of the molecular sieves on the structural, optical, thermal, mechanical, morphologic and protonic conductivity properties and water/methanol swelling degree of membranes were investigated.

  8. Immobilization to prevent enzyme incompatibility with proteases

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was s

  9. Cytochrome P450 enzyme systems in fungi

    Brink, H.M. van den; Gorcom, R.F.M. van; Hondel, C.A.M.J.J. van den; Punt, P.J.

    1998-01-01

    The involvement of cytochrome P450 enzymes in many complex fungal bioconversion processes has been characterized in recent years. Accordingly, there is now considerable scientific interest in fungal cytochrome P450 enzyme systems. In contrast to S. cerevisiae, where surprisingly few P450 genes have

  10. Lemon Polyphenols Suppress Diet-induced Obesity by Up-Regulation of mRNA Levels of the Enzymes Involved in β-Oxidation in Mouse White Adipose Tissue

    Fukuchi, Yoshiko; Hiramitsu, Masanori; Okada, Miki; Hayashi, Sanae; Nabeno, Yuka; Osawa, Toshihiko; Naito, Michitaka

    2008-01-01

    The aim of this study was to investigate the effect of dietary lemon polyphenols on high-fat diet-induced obesity in mice, and on the regulation of the expression of the genes involved in lipid metabolism to elucidate the mechanisms. Mice were divided into three groups and fed either a low fat diet (LF) or a high fat diet (HF) or a high fat diet supplemented with 0.5% w/w lemon polyphenols (LP) extracted from lemon peel for 12 weeks. Body weight gain, fat pad accumulation, the development of ...

  11. The ENZYME data bank.

    Bairoch, A

    1994-01-01

    The ENZYME data bank is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) and it contains the following data for each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided: EC number Recommended name Alternative names (if any) Catalytic activity Cofactors (if any) Pointers to the SWISS-PROT protein sequence entrie(s) that correspond to the enzyme (if any) Pointers to human disease(s) associated with a deficiency of the enzyme (if any). PMID:7937072

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

    杨栋; 万立华; 涂政; 石屹; 胡兰

    2014-01-01

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

  13. Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and design to avoid CYP3A4 time-dependent inhibition.

    Igawa, Hideyuki; Takahashi, Masashi; Shirasaki, Mikio; Kakegawa, Keiko; Kina, Asato; Ikoma, Minoru; Aida, Jumpei; Yasuma, Tsuneo; Okuda, Shoki; Kawata, Yayoi; Noguchi, Toshihiro; Yamamoto, Syunsuke; Fujioka, Yasushi; Kundu, Mrinalkanti; Khamrai, Uttam; Nakayama, Masaharu; Nagisa, Yasutaka; Kasai, Shizuo; Maekawa, Tsuyoshi

    2016-06-01

    Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents, and numerous drug discovery programs are dedicated to finding small-molecule MCH receptor 1 (MCHR1) antagonists. We recently reported novel pyridine-2(1H)-ones as aliphatic amine-free MCHR1 antagonists that structurally featured an imidazo[1,2-a]pyridine-based bicyclic motif. To investigate imidazopyridine variants with lower basicity and less potential to inhibit cytochrome P450 3A4 (CYP3A4), we designed pyridine-2(1H)-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a 1H-benzimidazole motif showed comparable binding affinity to MCHR1 to the corresponding imidazopyridine derivative 1. Optimization of 6a afforded a series of potent thiophene derivatives (6q-u); however, most of these were found to cause time-dependent inhibition (TDI) of CYP3A4. As bioactivation of thiophenes to form sulfoxide or epoxide species was considered to be a major cause of CYP3A4 TDI, we introduced electron withdrawing groups on the thiophene and found that a CF3 group on the ring or a Cl adjacent to the sulfur atom helped prevent CYP3A4 TDI. Consequently, 4-[(5-chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s) was identified as a potent MCHR1 antagonist without the risk of CYP3A4 TDI, which exhibited a promising safety profile including low CYP3A4 inhibition and exerted significant antiobesity effects in diet-induced obese F344 rats. PMID:27112449

  14. Enzyme Therapy: Current Perspectives.

    UmaMaheswari, Thiyagamoorthy; Hemalatha, Thiagarajan; Sankaranarayanan, Palavesam; Puvanakrishnan, Rengarajulu

    2016-01-01

    Enzymes control all metabolic processes in human system from simple digestion of food to highly complex immune response. Physiological reactions occuring in healthy individuals are disturbed when enzymes are deficient or absent. Enzymes are administered for normalizing biological function in certain pathologies. Initially, crude proteolytic enzymes were used for the treatment of gastrointestinal disorders. Recent advances have enabled enzyme therapy as a promising tool in the treatment of cardiovascular, oncological and hereditary diseases. Now, a spectrum of other diseases are also covered under enzyme therapy. But, the available information on the use of enzymes as therapeutic agents for different diseases is scanty. This review details the enzymes which have been used to treat various diseases/disorders. PMID:26891548

  15. Involved Consumers and Advertising Involvement

    Lawlor, Katrina

    1988-01-01

    The question of consumer involvement has at times taken on the appearance of a theoretical quagmire. The proliferation of definitions apart, this confusion has been exacerbated by the failure to distinguish adequately between advertising and consumer involvement. The research outlined in this article attempts to probe the possible relationship between these two discrete entities. It takes as a starting point Kassarjian's postulate of a generalised trait of purchasing involvement. This novel ...

  16. Mediated effect of ultrasound treated Diclofenac on mussel hemocytes: First evidence for the involvement of respiratory burst enzymes in the induction of DCF-mediated unspecific mode of action.

    Toufexi, Eirini; Dailianis, Stefanos; Vlastos, Dimitris; Manariotis, Ioannis D

    2016-06-01

    The present study investigates the toxic behavior of diclofenac (DCF) before and after its ultrasound (US) treatment, as well as the involvement of intracellular target molecules, such as NADPH oxidase and NO synthase, in the DCF-induced adverse effects on hemocytes of mussel Mytilus galloprovincialis. In this context, appropriate volumes (350 and 500mL) of DCF solutions (at concentrations of 2, 2.5, 5 and 10mgL(-1)) were treated under different ultrasound operating conditions (frequency at 582 and 862kHz, electric power density at 133 and 167W) for assessing US method efficiency. In parallel, DCF and US DCF-mediated cytotoxic (in terms of cell viability measured with the use of neutral red uptake/NRU method), oxidative (in terms of superoxide anions/(.)O2(-), nitric oxides such as NO2(-) and lipid peroxidation products, such as malondialdehyde/MDA content) and genotoxic (DNA damage measured by the use of Comet assay method) effects were investigated in hemocytes exposed for 1h to 5, 10 and 100ngL(-1) and 1, 10 and 20μgL(-1) of DCF. The involvement of NADPH oxidase and NO synthase to the DCF-induced toxicity was further investigated by the use of 10μΜ L-NAME, a NO synthase inhibitor and 10μΜ DPI, a NADPH oxidase inhibitor. According to the results, 350mL of 2mgL(-1) DCF showed higher degradation (>50%) under 167W electric power density and frequency at 862kHz for 120min, compared to degradation in all other cases, followed by a significant elimination of its toxicity. Specifically, US DCF-treated hemocytes showed a significant attenuation of DCF-mediated cytotoxic, oxidative and genotoxic effects, which appeared to be caused by NADPH oxidase and NO synthase activation, since their inhibition was followed by a significant elimination of (.)O2(-) and NO2(-) generation and the concomitant oxidative damage within cells. The results of the present study showed for the first time that unspecific mode of action of DCF, associated with the induction of NADPH oxidase

  17. Enzyme inhibition by iminosugars

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus;

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes with...

  18. Intestinal CYP3A4 protects against lithocholic acid-induced hepatotoxicity in intestine-specific VDR-deficient mice[S

    Cheng, Jie; Fang, Zhong-Ze; Kim, Jung-Hwan; Krausz, Kristopher W.; Tanaka, Naoki; Chiang, John Y. L.; Frank J. Gonzalez

    2014-01-01

    Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (VdrΔIEpC) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytoch...

  19. Expression of two drug-metabolizing cytochrome P450-enzymes in human salivary glands

    Kragelund, C; Hansen, C; Torpet, L A;

    2008-01-01

    OBJECTIVE: The oral cavity is constantly lubricated by saliva and even small amounts of xenobiotics and / or their metabolites in the saliva may affect the oral mucosa. Our aim was therefore to clarify if xenobiotic metabolizing enzymes CYP1A2 and CYP3A4 are expressed in salivary glands. METHODS...

  20. Deubiquitylating enzymes and disease

    Baker Rohan T

    2008-10-01

    Full Text Available Abstract Deubiquitylating enzymes (DUBs can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin, including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80–90 DUBs in five different subfamilies, only a handful of which have been characterized with respect to the proteins that they interact with and deubiquitylate. Several other DUBs have been implicated in various disease processes in which they are changed by mutation, have altered expression levels, and/or form part of regulatory complexes. Specific examples of DUB involvement in various diseases are presented. While no specific drugs targeting DUBs have yet been described, sufficient functional and structural information has accumulated in some cases to allow their rapid development. Publication history Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com.

  1. Enzymes for improved biomass conversion

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  2. Protein engineering of enzymes for process applications

    Woodley, John M

    2013-01-01

    Scientific progress in the field of enzyme modification today enables the opportunity to tune a given biocatalyst for a specific industrial application. Much work has been focused on extending the substrate repertoire and altering selectivity. Nevertheless, it is clear that many new forthcoming...... opportunities will be targeted on modification to enable process application. This article discusses the challenges involved in enzyme modification focused on process requirements, such as the need to fulfill reaction thermodynamics, specific activity under the required conditions, kinetics at required...

  3. Enzymes and muscle diseases

    M. Plebani

    2011-09-01

    Full Text Available Skeletal muscle disorders may result in release of muscle enzymes into the circulation and give increased serum enzyme activity. A variety of enzymes routinely determined in the clinical laboratory may be elevated, but creatine kinase is the enzyme present in the highest concentration in muscle, and in every variety of muscle disease is the serum enzyme which shows the greatest incidence and degree of elevation. Aspartate aminotransferase is the enzyme associated most significantly with inflammation. A diagnostic algorithm based on the combined measurement of creatine kinase, aspartate aminotransferase and aldolase has been found to discriminate muscular distrophies from polymyositis and other myopathies. This combination of laboratory tests has diagnostic application and thus allows the clinician to better select patients who need to have a skeletal muscle biopsy as a diagnostic procedure.

  4. Modeling amperometric biosensors based on allosteric enzymes

    Liutauras Ričkus

    2013-09-01

    Full Text Available Computational modeling of a biosensor with allosteric enzyme layer was investigated in this study. The operation of the biosensor is modeled using non-stationary reaction-diffusion equations. The model involves three regions: the allosteric enzyme layer where the allosteric enzyme reactions as well as then mass transport by diffusion take place, the diffusion region where the mass transport by diffusion and non-enzymatic reactions take place and the convective region in which the analyte concentration is maintained constant. The biosensor response on dependency substrate concentration, cooperativity coefficient and the diffusion layer thickness on the same parameters have been studied.

  5. Activation of interfacial enzymes at membrane surfaces

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi;

    2006-01-01

    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s......PLA2), are only activated at the interface between water and membrane surfaces, where they lead to a break-down of the lipid molecules into lysolipids and free fatty acids. The activation is critically dependent on the physical properties of the lipid-membrane substrate. A topical review is given of...

  6. 1, 3-Dipolar cycloaddition reactions: Synthesis of 5-benzyl-1-(2',4'-dibromophenyl)-3-(4"-substituted phenyl)-3a,4,6,6a-tetrahydro-1, 5-pyrrolo[3,4-]pyrazole-4,6-dione derivatives

    Manpreet Kaur; Baldev Singh; Baljit Singh

    2013-11-01

    1,3-Dipolar cycloaddition of nitrilimines 3 with -benzyl maleimide 4 has provided 5-benzyl-1-(2',4'-dibromophenyl)-3-(4"-substituted phenyl)-3a,4,6,6a-tetrahydro-1,5-pyrrolo[3,4-]pyrazole-4,6-dione derivatives 5 in excellent yield as the only isomer through a concerted pathway.

  7. Hyperconjugation with lone pair of morpholine nitrogen stabilizes transition state for phenyl hydroxylation in CYP3A4 metabolism of ( S)- N-[1-(3-morpholin-4-yl phenyl) ethyl]-3-phenylacrylamide

    Shaikh, Abdul Rajjak; Broclawik, Ewa; Ismael, Mohamed; Tsuboi, Hideyuki; Koyama, Michihisa; Kubo, Momoji; Del Carpio, Carlos A.; Miyamoto, Akira

    2006-02-01

    Using quantum chemical modelling we describe a novel effect in the mechanism of CYP3A4 metabolism for the arene substrate with o-substituent yielding a lone pair donation to conjugate π system; this will compensate for the loss of aromaticity on formation of the tetrahedral complex and lower the rate-determining energy barrier.

  8. Enzymes and Genes Involved in Aerobic Alkane Degradation

    ZongzeShao

    2013-05-01

    Full Text Available Alkanes are major constituents of crude oil. They are also present at low concentrations in diverse non-contaminated because many living organisms produce them as chemo-attractants or as protecting agents against water loss. Alkane degradation is a widespread phenomenon in nature. The numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing alkanes as a carbon and energy source, have been isolated and characterized. This review summarizes the current knowledge of how bacteria metabolize alkanes aerobically, with a particular emphasis on the oxidation of long-chain alkanes, including factors that are responsible for chemotaxis to alkanes , transport across cell membrane of alkanes , the regulation of alkane degradation gene and initial oxidation.

  9. Characterization of Carbohydrate Active Enzymes Involved in Arabinogalactan Protein Metabolism

    Knoch, Eva

    accounting for up to 90% of the total mass. The glycan contains mostly galactose and arabinose, but glucoronic acid and other less-abundant sugars like rhamnose, xylose and fucose are also found. Although AGPs are important for normal plant growth and are found throughout the plant in virtually all organs...

  10. Magnetically responsive enzyme powders

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction

  11. HYDRATION AND ENZYME ACTIVITY

    Poole, P.

    1984-01-01

    Hydration induced conformation and dynamic changes are followed using a variety of experimental techniques applied to hen egg white lysozyme. These changes are completed just before the onset of enzyme activity, which occurs before all polar groups are hydrated, and before monolayer coverage is attained. We suggest that these hydration induced changes are necessary for the return of enzyme activity.

  12. Directed Evolution of Enzymes

    Doucet, Nicolas; Pelletier, Joelle,

    2004-01-01

    This brief technological report presents an overview of techniques and applications in the field of directed evolution of enzyme catalysts. These techniques allow for the creation of modified enzymes that are better adapted to many industrial contexts. Recent applications in organic synthesis as well as commercial, biomedical, and environmental usage of these modified catalysts will be presented.

  13. Magnetically responsive enzyme powders

    Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2015-04-15

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  14. Artificial Enzymes, "Chemzymes"

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M;

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that...... successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well as...

  15. Comparative gene expression of intestinal metabolizing enzymes.

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs. PMID:19746353

  16. Enzymes in Analytical Chemistry.

    Fishman, Myer M.

    1980-01-01

    Presents tabular information concerning recent research in the field of enzymes in analytic chemistry, with methods, substrate or reaction catalyzed, assay, comments and references listed. The table refers to 128 references. Also listed are 13 general citations. (CS)

  17. Membrane Assisted Enzyme Fractionation

    Yuan, Linfeng

    difference. In this thesis, separations using crossflow elecro-membrane filtration (EMF) of amino acids, bovine serum albumin (BSA) and industrial enzymes from Novozymes were performed. The main objective of this study was to investigate the technological feasibility of EMF in the application of industrial...... enzyme fractionation, such as removal of a side activity from the main enzyme activity. As a proof-of-concept, amino acids were used as model solution to test the feasibility of EMF in the application of amphoteric molecule separation. A single amino acid was used to illustrate the effect of an electric...... TMP on the separation performance were very small in the investigated range. The mass transport of each enzyme can be well explained by the Extended-Nernst-Planck equation. Better separation was observed at lower feed concentration, higher solution pH in the investigated range and with a polysulfone...

  18. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  19. Enzyme with rhamnogalacturonase activity.

    Kofod, L.V.; Andersen, L N; Dalboge, H; Kauppinen, M.S.; Christgau, S; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A. G. J.; Schols, H.A.

    1998-01-01

    An enzyme exhibiting rhamnogalacturonase activity, capable of cleaving a rhamnogalacturonan backbone in such a manner that galacturonic acids are left as the non-reducing ends, and which exhibits activity on hairy regions from a soy bean material and/or on saponified hairy regions from a sugar beet material. The enzyme has the amino acid sequence of SEQ ID NO:2 and is encoded by the DNA sequence of SEQ ID NO:1

  20. Overproduction of ligninolytic enzymes

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  1. Deubiquitylating enzymes and their emerging role in plant biology

    Isono, Erika; Nagel, Marie-Kristin

    2014-01-01

    Ubiquitylation is a reversible post-translational modification that is involved in various cellular pathways and that thereby regulates various aspects of plant biology. For a long time, functional studies of ubiquitylation have focused on the function of ubiquitylating enzymes, especially the E3 ligases, rather than deubiquitylating enzymes (DUBs) or ubiquitin isopeptidases, enzymes that hydrolyze ubiquitin chains. One reason may be the smaller number of DUBs in comparison to E3 ligases, imp...

  2. Measuring Solution Viscosity and its Effect on Enzyme Activity

    Uribe Salvador; Sampedro José G.

    2003-01-01

    In proteins, some processes require conformational changes involving structural domain diffusion. Among these processes are protein folding, unfolding and enzyme catalysis. During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle. According to Kramers theory, solvent viscosity results in friction against proteins in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes. Solution viscosity was increas...

  3. On the Structural Context and Identification of Enzyme Catalytic Residues

    Yu-Tung Chien; Shao-Wei Huang

    2013-01-01

    Enzymes play important roles in most of the biological processes. Although only a small fraction of residues are directly involved in catalytic reactions, these catalytic residues are the most crucial parts in enzymes. The study of the fundamental and unique features of catalytic residues benefits the understanding of enzyme functions and catalytic mechanisms. In this work, we analyze the structural context of catalytic residues based on theoretical and experimental structure flexibility. The...

  4. Charge-transport-mediated recruitment of DNA repair enzymes

    Fok, Pak-Wing; Guo, Chin-Lin; Chou, Tom

    2008-01-01

    Damaged or mismatched bases in DNA can be repaired by base excision repair enzymes (BER) that replace the defective base. Although the detailed molecular structures of many BER enzymes are known, how they colocalize to lesions remains unclear. One hypothesis involves charge transport (CT) along DNA [Yavin et al., Proc. Natl. Acad. Sci. U.S.A. 102, 3546 (2005)]. In this CT mechanism, electrons are released by recently adsorbed BER enzymes and travel along the DNA. The electrons can scatter (by...

  5. Differential modulation of the expression of important drug metabolising enzymes and transporters by endothelin-1 receptor antagonists ambrisentan and bosentan in vitro.

    Weiss, Johanna; Herzog, Melanie; Haefeli, Walter Emil

    2011-06-25

    The safety and effectiveness of drugs used to treat chronic diseases critically depend on their propensity to interact with co-administered drugs. Induction of enzymes and drug transporters involved in the clearance and distribution of drugs may critically reduce exposure with their substrates and thus lead to nonresponse. We therefore investigated the impact of the endothelin-1 receptor antagonists bosentan and ambrisentan on the expression of relevant human efflux and uptake transporters and on phase 1 and phase 2 enzymes. LS180 adenocarcinoma cells were treated for four days with bosentan or ambrisentan (1-50 μM), the positive control rifampicin, or medium only (negative control). For evaluation of bosentan also HuH-7 human hepatoma cells were used and treated similarly. Gene expression was quantified at the mRNA level by real-time reverse transcription polymerase chain reaction and for some genes also at the protein level by western blot analysis. Comparable to rifampicin, bosentan was a moderate to strong inductor for all cytochrome P450 isozymes and ATP-binding cassette transporters tested, and it also induced organic anion transporting polypeptides. 50 μM bosentan up-regulated e.g. CYP3A4 8.5-fold, ABCB1 5.1-fold, and ABCB11 1.9-fold at the mRNA level in LS180 cells. In HuH-7 cells induction was much less pronounced (e.g. CYP3A4 1.9-fold for bosentan). In contrast, ambrisentan only weakly induced some of the genes investigated in LS180 cells. These findings corroborate the in vivo finding that bosentan is much more prone to drug interactions than ambrisentan. PMID:21501604

  6. Enzyme Technology: A Practical Topic in Basic Chemical Education.

    Grunwald, Peter

    1986-01-01

    Discusses the importance of teaching about enzymes in chemistry. Mentions several applications of enzyme technology to other fields. Describes an experiment involving the immobilization of yeast cells as a biocatalyst for the ethanol production from glucose. Argues for more biotechnology to be integrated into basic chemistry courses. (TW)

  7. Pharmacokinetics of Lidocaine Hydrochloride Metabolized by CYP3A4 in Chinese Han Volunteers Living at Low Altitude and in Native Han and Tibetan Chinese Volunteers Living at High Altitude.

    Zhang, Juanling; Zhu, Junbo; Yao, Xingchen; Duan, Yabin; Zhou, Xuejiao; Yang, Meng; Li, Xiangyang

    2016-01-01

    To investigate the pharmacokinetics of lidocaine hydrochloride metabolized by cytochrome P450 3A4 (CYP3A4) in Chinese Han volunteers living at low altitude (LA) and in native Han and Tibetan Chinese volunteers living at high altitude, lidocaine hydrochloride 10 mg was given by intramuscular injection to 3 groups: Han volunteers living at LA, and native Han and Tibetan volunteers living at a high altitude. Blood samples were collected before the (baseline) study drug was given and at 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0 h after study drug administration. Lidocaine hydrochloride in plasma was determined by RP-HPLC. Pharmacokinetics parameters of lidocaine hydrochloride showed that there were no significant difference between the native Han and Tibetan volunteers, but the t1/2 was 29.8 and 29.8% higher in 2 groups, respectively, than in the LA group. To study related mechanism, the effects of exposure to chronic high-altitude hypoxia (CHH) on the activity and expression of CYP3A1 were examined in rats. Rats were divided into LA, chronic moderate altitude hypoxia, and CHH groups. CHH caused significant decreases in the activity and protein and mRNA expression of rat CYP3A1 in vivo. This study found significant changes in the disposition of lidocaine hydrochloride in native healthy Tibetan and Han Chinese subjects living at a high altitude in comparison to healthy Han Chinese subjects living at LA, it might be due to significant decreases in the activity and protein and mRNA expression of CYP3A4 under CHH condition. PMID:26730802

  8. Angiotensin-converting enzyme

    Sørensen, P G; Rømer, F K; Cortes, D

    1984-01-01

    In order to evaluate bleomycin-associated lung damage in humans, lung function parameters and serum levels of the endothelial-bound angiotensin-converting enzyme (ACE) were determined by serial measurements in 11 patients who were treated for testicular cancer. None developed clinical or radiolog......In order to evaluate bleomycin-associated lung damage in humans, lung function parameters and serum levels of the endothelial-bound angiotensin-converting enzyme (ACE) were determined by serial measurements in 11 patients who were treated for testicular cancer. None developed clinical or...

  9. Hyperthermophilic Enzymes with Industrial Applications

    Mojsov, Kiro; Janevski, Aco; Andronikov, Darko; Zezova, Silvana

    2014-01-01

    Hyperthermophilic enzymes are typically thermostable and are optimally active at high temperatures. Hyperthermophilic enzymes are very similar to their mesophilic homologues. No single mechanism that is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermo stability must be found in a small number of highly specific alterations. In this review are described current uses and potential applications of thermophilic and hyperthermophilic enzymes as ...

  10. The ENZYME database in 2000.

    Bairoch, A

    2000-01-01

    The ENZYME database is a repository of information related to the nomenclature of enzymes. In recent years it has became an indispensable resource for the development of metabolic databases. The current version contains information on 3705 enzymes. It is available through the ExPASy WWW server (http://www.expasy.ch/enzyme/ ). PMID:10592255

  11. The surface science of enzymes

    Rod, Thomas Holm; Nørskov, Jens Kehlet

    2002-01-01

    One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function? To...... solve these problems we must understand in some detail how enzymes interact with reactants from its surroundings. These interactions take place at the surface of the enzyme and the question of enzyme function can be viewed as the surface science of enzymes. In this article we discuss how to describe...... catalysis by enzymes, and in particular the analogies between enzyme catalyzed reactions and surface catalyzed reactions. We do this by discussing two concrete examples of reactions catalyzed both in nature (by enzymes) and in industrial reactors (by inorganic materials), and show that although analogies...

  12. Magnetically responsive enzyme powders

    Pospišková, K.; Šafařík, Ivo

    2015-01-01

    Roč. 380, APR 2015 (2015), s. 197-200. ISSN 0304-8853 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : enzyme powders * cross-linking * magnetic modification * magnetic separation * magnetic iron oxides particles * microwave-assisted synthesis Subject RIV: CE - Biochemistry Impact factor: 1.970, year: 2014

  13. Enzymes in Forest Soils

    Baldrian, Petr; Štursová, Martina

    Heidelberg, Dordrecht, NY: Springer, 2011 - (Shukla, G.; Varma, A.), s. 61-73 ISBN 978-3-642-14225-3 R&D Projects: GA ČR GA526/08/0751; GA MŠk OC08050 Institutional research plan: CEZ:AV0Z50200510 Keywords : forest soils * soil ecology * enzymes Subject RIV: EE - Microbiology, Virology

  14. Enzymes of Saprotrophic Basidiomycetes

    Baldrian, Petr

    Amsterdam: Academic Press, 2007, s. 19-41. ISBN 978-0-12-374185-1 R&D Projects: GA AV ČR KJB600200516; GA ČR GA526/05/0168; GA MŠk LC06066 Institutional research plan: CEZ:AV0Z50200510 Keywords : saprotrophic basidiomycetes * extracellular enzymes * polymers Subject RIV: EE - Microbiology, Virology

  15. Computational enzyme design

    Bolon, Daniel N.

    2002-08-01

    The long-term objective of computational enzyme design is the ability to generate efficient protein catalysts for any chemical reaction. This thesis develops and experimentally validates a general computational approach for the design of enzymes with novel function. In order to include catalytic mechanism in protein design, a high-energy state (HES) rotamer (side chain representation) was constructed. In this rotamer, substrate atoms are in a HES. In addition, at least one amino acid side chain is positioned to interact favorably with substrate atoms in their HES and facilitate the reaction. Including an amino acid side chain in the HES rotamer automatically positions substrate relative to a protein scaffold and allows protein design algorithms to search for sequences capable of interacting favorably with the substrate. Because chemical similarity exists between the transition state and the high-energy state, optimizing the protein sequence to interact favorably with the HES rotamer should lead to transition state stabilization. In addition, the HES rotamer model focuses the subsequent computational active site design on a relevant phase space where an amino acid is capable of interacting in a catalytically active geometry with substrate. Using a HES rotamer model of the histidine mediated nucleophilic hydrolysis of p-nitrophenyl acetate, the catalytically inert 108 residue E. coli thioredoxin as a scaffold, and the ORBIT protein design software to compute sequences, an active site scan identified two promising active site designs. Experimentally, both candidate ?protozymes? demonstrated catalytic activity significantly above background. In addition, the rate enhancement of one of these ?protozymes? was the same order of magnitude as the first catalytic antibodies. Because polar groups are frequently buried at enzyme-substrate interfaces, improved modeling of buried polar interactions may benefit enzyme design. By studying native protein structures, rules have been

  16. Plant Proteases Involved in Regulated Cell Death.

    Zamyatnin, A A

    2015-12-01

    Each plant genome encodes hundreds of proteolytic enzymes. These enzymes can be divided into five distinct classes: cysteine-, serine-, aspartic-, threonine-, and metalloproteinases. Despite the differences in their structural properties and activities, members of all of these classes in plants are involved in the processes of regulated cell death - a basic feature of eukaryotic organisms. Regulated cell death in plants is an indispensable mechanism supporting plant development, survival, stress responses, and defense against pathogens. This review summarizes recent advances in studies of plant proteolytic enzymes functioning in the initiation and execution of distinct types of regulated cell death. PMID:26878575

  17. INFLUENCE OF THE CYP3A4 ISOENZYME METABOLIC ACTIVITY AND CYP2C19 GENE POLYMORPHISMS ON CLOPIDOGREL ANTIPLATELET EFFECT IN PATIENTS WITH ACUTE CORONARY SYNDROME UNDERGOING PERCUTANEOUS CORONARY INTERVENTION

    K. B. Mirzaev

    2015-09-01

    Full Text Available Aim. Carriership of CYP2C19*2 allelic variant and reduced CYP3A4 activity can affect the formation of clopidogrel’s active metabolite and, respectively, its antiplatelet effect. We sought to determine the impact of CYP3A4 isoenzyme activity and CYP2C19 polymorphisms on platelet aggregation.Material and methods. The study included 81 patients with acute coronary syndrome (ACS and subsequent percutaneous coronary intervention (PCI: 64 males and 17 females, mean age 63.9±10.9 years. CYP2C19 allelic variants were detected by the method of real-time polymerase chain reaction. CYP3A4 isoenzyme activity was estimated by urinary 6-β-hydroxycortisol/free cortisol ratio (6-OHC/FC using the method of high-performance liquid chromatography. Platelet functional activity was evaluated by a portative aggregometer - the VerifyNow P2Y12 assay.Results. Logistic regression analysis has demonstrated significantly increased risk of clopidogrel resistance in patients-carriers of the CYP2C19*2 polymorphism (p=0.022. CYP2C19*2 non-carriers had significantly higher mean platelet inhibition percentage as compared with the carriers of this allele: 30.7±20.1 in the CYP2C19*1/*1 group vs 18.2±16.4 in the CYP2C19*1/*2 one (р=0.03. Clopidogrel laboratory resistance (P2Y12 Reaction Units (PRU>208 was found out to be higher in the CYP2C19*2-carriers as compared with non-carriers: 53.8% in the patients with the CYP2C19*1/*2 genotype and 16.2% in subjects with the CYP2C19*1/*1 genotype (odds ratio [OR]=1.8; 95% confidence interval [95% CI]: 1.0–3.2; р=0.0067. Linear regression analysis has revealed that smaller mean diameter of stent slightly reduces the risk of clopidogrel resistance development. No significant distinctions in urinary 6-OHC/FC ratios (the marker of CYP3A4 activity were observed: 3.4±2.8 in the PRU>208 group and 3.2±3.0 in the PRU<208 group (p=0.8. Besides, no significant correlation between platelet activity and the 6-OHC/FC ratio was found (р=0

  18. Recent advances in rational approaches for enzyme engineering

    Kerstin Steiner

    2012-09-01

    Full Text Available Enzymes are an attractive alternative in the asymmetric syntheses of chiral building blocks. To meet the requirements of industrial biotechnology and to introduce new functionalities, the enzymes need to be optimized by protein engineering. This article specifically reviews rational approaches for enzyme engineering and de novo enzyme design involving structure-based approaches developed in recent years for improvement of the enzymes’ performance, broadened substrate range, and creation of novel functionalities to obtain products with high added value for industrial applications.

  19. Perspectives of Solid State Fermentation for Production of Food Enzymes

    Cristobal Noe Aguilar

    2008-01-01

    Full Text Available Food industry represents one of the economic sectors where microbial metabolites have found a wide variety of applications. This is the case of some enzymes, such as amylases, cellulases, pectinases and proteases which have played a very important role as food additives. Most of these enzymes have been produced by submerged cultures at industrial level. Many works in the literature present detailed aspects involved with those enzymes and their importance in the food industry. However, the production and application studies of those enzymes produced by solid state fermentations are scarce in comparison with submerged fermentation. This review involves production aspects of the seven enzymes: tannases, pectinases, caffeinases, mannanases, phytases, xylanases and proteases, which can be produced by solid state fermentation showing attractive advantages. Additionally, process characteristics of solid state fermentation are considered.

  20. Genetic variation in the 3'-UTR of CYP1A2, CYP2B6, CYP2D6, CYP3A4, NR1I2 and UGT2B7: potential effect on regulation by microRNA and pharmacogenomics relevance

    Marelize eSwart

    2014-06-01

    Full Text Available Introduction: Pharmacogenomics research has concentrated on variation in genes coding for drug metabolising enzymes, transporters and nuclear receptors. However, variation affecting microRNA could also play a role in drug response. This project set out to investigate potential microRNA target sites in 11 genes and the extent of variation in the 3'-UTR of six selected genes; CYP1A2, CYP2B6, CYP2D6, CYP3A4, NR1I2 and UGT2B7. Methods: Fifteen microRNA target prediction algorithms were used to identify microRNAs predicted to regulate 11 genes. The 3'-UTR of the 6 genes which topped the list of potential microRNA targets was sequenced in 30 black South Africans. In addition, genetic variants within these genes were investigated for interference with mRNA-microRNA interactions. Potential effects of observed variants were determined using in silico prediction tools. Results: The 11 genes coding for DMEs, transporters and nuclear receptors were predicted to be targets of microRNAs with CYP2B6, NR1I2 (PXR, CYP3A4 and CYP1A2, interacting with the most microRNAs. The majority of identified genetic variants were predicted to interfere with microRNA regulation. For example, the variant, rs1054190C in NR1I2 was predicted to result in the presence of a binding site for the microRNA miR-1250-5p, while the variant rs1054191G was predicted to result in the absence of a recognition site for miR-371b-3p, miR-4258 and miR-4707-3p. Fifteen of the seventeen, novel variants occurred within microRNA target sequences.Conclusion: The 3'-UTR harbours variation that is likely to influence regulation of specific genes by microRNA. In silico prediction followed by functional validation could aid in decoding the contribution of variation in the 3'-UTR, to some unexplained heritability that affects drug response. Understanding the specific role of each microRNA may lead to identification of markers for targeted therapy and therefore improve personalized drug treatment.

  1. Non-homologous isofunctional enzymes: A systematic analysis of alternative solutions in enzyme evolution

    Wolf Yuri I

    2010-04-01

    Full Text Available Abstract Background Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (nonhomologous relationships between proteins. Results We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. Conclusions These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity. Reviewers This article was reviewed by Andrei

  2. Deubiquitylating enzymes and disease

    Baker Rohan T; Taylor Matthew C; Singhal Shweta

    2008-01-01

    Abstract Deubiquitylating enzymes (DUBs) can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin), including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80–90 DUBs in five different subfamilies, only a handful of which have been charac...

  3. Linking hydrolysis performance to Trichoderma reesei cellulolytic enzyme profile.

    Lehmann, Linda; Rønnest, Nanna P; Jørgensen, Christian I; Olsson, Lisbeth; Stocks, Stuart M; Jørgensen, Henrik S; Hobley, Timothy

    2016-05-01

    Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, for example, by spiking with single enzymes and monitoring hydrolysis performance. In this study, a multivariate approach, partial least squares regression, was used to see whether it could help explain the correlation between enzyme profile and hydrolysis performance. Diverse enzyme mixtures were produced by T. reesei Rut-C30 by exploiting various fermentation conditions and used for hydrolysis of washed pretreated corn stover as a measure of enzyme performance. In addition, the enzyme mixtures were analyzed by liquid chromatography-tandem mass spectrometry to identify and quantify the different proteins. A multivariate model was applied for the prediction of enzyme performance based on the combination of different proteins present in an enzyme mixture. The multivariate model was used for identification of candidate proteins that are correlated to enzyme performance on pretreated corn stover. A very large variation in hydrolysis performance was observed and this was clearly caused by the difference in fermentation conditions. Besides β-glucosidase, the multivariate model identified several xylanases, Cip1 and Cip2, as relevant proteins to study further. Biotechnol. Bioeng. 2016;113: 1001-1010. © 2015 Wiley Periodicals, Inc. PMID:26524197

  4. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  5. Enzyme Molar Fractions: A Powerful Tool for Understanding Enzyme Kinetics.

    Serra, Juan L.; And Others

    1986-01-01

    Deduces the relationship between reduced velocity and molar fractions for productive enzyme complexes; obtains the mathematical expression of molar fractions for an enzyme with two specific binding sites per molecule; and proposes a useful plot to follow the dependence of enzyme molar fractions with the concentration of one of its ligands. (JN)

  6. Enzymes for Enhanced Oil Recovery (EOR)

    Nasiri, Hamidreza

    2011-04-15

    Primary oil recovery by reservoir pressure depletion and secondary oil recovery by waterflooding usually result in poor displacement efficiency. As a consequence there is always some trapped oil remaining in oil reservoirs. Oil entrapment is a result of complex interactions between viscous, gravity and capillary forces. Improving recovery from hydrocarbon fields typically involves altering the relative importance of the viscous and capillary forces. The potential of many EOR methods depends on their influence on fluid/rock interactions related to wettability and fluid/fluid interactions reflected in IFT. If the method has the potential to change the interactions favorably, it may be considered for further investigation, i.e. core flooding experiment, pilot and reservoir implementation. Enzyme-proteins can be introduced as an enhanced oil recovery method to improve waterflood performance by affecting interactions at the oil-water-rock interfaces. An important part of this thesis was to investigate how selected enzymes may influence wettability and capillary forces in a crude oil-brine-rock system, and thus possibly contribute to enhanced oil recovery. To investigate further by which mechanisms selected enzyme-proteins may contribute to enhance oil recovery, groups of enzymes with different properties and catalytic functions, known to be interfacially active, were chosen to cover a wide range of possible effects. These groups include (1) Greenzyme (GZ) which is a commercial EOR enzyme and consists of enzymes and stabilizers (surfactants), (2) The Zonase group consists of two types of pure enzyme, Zonase1 and Zonase2 which are protease enzymes and whose catalytic functions are to hydrolyze (breakdown) peptide bonds, (3) The Novozyme (NZ) group consists of three types of pure enzyme, NZ2, NZ3 and NZ6 which are esterase enzymes and whose catalytic functions are to hydrolyze ester bonds, and (4) Alpha-Lactalbumin ( -La) which is an important whey protein. The effect of

  7. Treating Wastewater With Immobilized Enzymes

    Jolly, Clifford D.

    1991-01-01

    Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

  8. Characterization of cytochrome P450 isoforms involved in sequential two-step bioactivation of diclofenac to reactive p-benzoquinone imines.

    den Braver, Michiel W; den Braver-Sewradj, Shalenie P; Vermeulen, Nico P E; Commandeur, Jan N M

    2016-06-24

    Idiosyncratic drug-induced lever injury (IDILI) is a rare but severe side effect of diclofenac (DF). Several mechanisms have been proposed as cause of DF-induced toxicity including the formation of protein-reactive diclofenac-1',4'-quinone imine (DF-1',4'-QI) and diclofenac-2,5-quinone imine (DF-2,5-QI). Formation of these p-benzoquinone imines result from two-step oxidative metabolism involving aromatic hydroxylation to 4'-hydroxydiclofenac and 5-hydroxydiclofenac followed by dehydrogenation to DF-1',4'-QI and DF-2,5-QI, respectively. Although the contribution of individual cytochrome P450s (CYPs) in aromatic hydroxylation of DF is well studied, the enzymes involved in the dehydrogenation reactions have been poorly characterized. The results of the present study show that both formation of 4'-hydroxydiclofenac and it subsequent bioactivation to DF-1',4'-QI is selectively catalyzed by CYP2C9. However, the two-step bioactivation to DF-2,5-QI appears to be catalyzed with highest activity by two different CYPs: 5-hydroxylation of DF is predominantly catalyzed by CYP3A4, whereas its subsequent bioactivation to DF-2,5-QI is catalyzed with 14-fold higher intrinsic clearance by CYP2C9. The fact that both CYPs involved in two-step bioactivation of DF show large interindividual variability may play a role in different susceptibility of patients to DF-induced IDILI. Furthermore, expression levels of these enzymes and protective enzymes might be important factors determining sensitivity of in vitro models for hepatotoxicity. PMID:27130197

  9. Linking Hydrolysis Performance to Trichoderma reesei Cellulolytic Enzyme Profile

    Lehmann, Linda Olkjær; Petersen, Nanna; I. Jørgensen, Christian;

    2016-01-01

    Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, e.g. by spiking with single enzymes and monitoring hydrolysis performance. In this study a multivariate...

  10. Liver involvement in Langerhans cell histiocytosis

    Liver involvement in Langerhans cell histiocytosis (LCH) typically presents with hepatomegaly and other signs of liver dysfunction. We present an 11-month-old child having only minimally elevated liver enzymes as an indication of liver involvement. Using sonography as the initial diagnostic tool followed by MRI, LCH of the liver was revealed. A review of sonographic, CT, MRI and MR cholangiopancreatography findings in liver LCH is presented. We recommend that physicians consider sonography and MRI screening for liver involvement in patients with newly diagnosed LCH, as periportal involvement may be present with little or no liver function abnormality present, as in this patient. (orig.)

  11. Liver involvement in Langerhans cell histiocytosis

    Wong, Adelaine; Ortiz-Neira, Clara L.; Abou Reslan, Walid; Kaura, Deepak [Alberta Children' s Hospital, Department of Diagnostic Imaging, Calgary, Alberta (Canada); Sharon, Raphael; Anderson, Ronald [Alberta Children' s Hospital, Department of Oncology, Calgary, AB (Canada); Pinto-Rojas, Alfredo [Alberta Children' s Hospital, Department of Pathology, Calgary, AB (Canada)

    2006-10-15

    Liver involvement in Langerhans cell histiocytosis (LCH) typically presents with hepatomegaly and other signs of liver dysfunction. We present an 11-month-old child having only minimally elevated liver enzymes as an indication of liver involvement. Using sonography as the initial diagnostic tool followed by MRI, LCH of the liver was revealed. A review of sonographic, CT, MRI and MR cholangiopancreatography findings in liver LCH is presented. We recommend that physicians consider sonography and MRI screening for liver involvement in patients with newly diagnosed LCH, as periportal involvement may be present with little or no liver function abnormality present, as in this patient. (orig.)

  12. Enzyme hydration, activity and flexibility : A neutron scattering approach

    Kurkal-Siebert, V [University of Heidelberg; Finney, J.L. [University College, London; Daniel, R. M. [University of Waikato, New Zealand; Smith, Jeremy C [ORNL

    2006-01-01

    Recent measurements have demonstrated enzyme activity at hydrations as low as 3%. The question of whether the hydration-induced enzyme flexibility is important for activity is addressed by performing picosecond dynamic neutron scattering experiments on pig liver esterase powders at various temperatures as well as solutions. At all temperatures and hydrations investigated here, significant quasielastic scattering intensity is found in the protein, indicating the presence of anharmonic, diffusive motion. As the hydration increases a temperature-dependent dynamical transition appears and strengthens involving additional diffusive motion. At low temperature, increasing hydration resulted in lower flexibility of the enzyme. At higher temperatures, systems containing sufficient number of water molecules interacting with the protein exhibit increased flexibility. The implication of these results is that, although the additional hydration-induced diffusive motion and flexibility at high temperatures in the enzyme detected here may be related to increased activity, they are not required for the enzyme to function.

  13. Synthesis and Crystal Structure of(3S,4R,Z)-3,6-dimethyl-2-(3-methylbut-2-enylidene)-2,3,3a,4,7,7a-hexahydrobenzofuran-3,4-diol

    CHEN Lei; FANG Hu-Biao; HUANG Nian-Yu; WANG Jun-Zhi; ZOU Kun

    2011-01-01

    The title compound of(3S,4R,Z)-3,6-dimethyl-2-(3-methylbut-2-enylidene)-2,3,3a,4,7,7a-hexahydrobenzofuran-3,4-diol,C15H22O3,as a potential gastric cytoprotective agent has been synthesized by the reduction of bisabolangelone in methanol with sodium borohydride.The title compound was characterized by IR and NMR spectra.Meanwhile,the crystal was obtained and determined by X-ray single-crystal diffraction.Crystal data:monoclinic system,space group P21 with a = 6.0692(12),b = 8.9954(18),c = 13.182(3) ,β = 92.59(3)°,V = 718.9(2) 3,Z = 2,F(000) = 272,Dc = 1.156 g/cm3,μ = 0.633 mm-1,R = 0.0362 and wR = 0.1051 for 9490 independent reflections(Rint = 0.0172) and 2461 observed reflections(I 2σ(I)).Intermolecular O-H…O interactions link the molecules into one-dimensional infinite chains running along the b axis,which contributes to the stability of the crystal structure.

  14. Predicting the "First dose in children" of CYP3A-metabolized drugs: Evaluation of scaling approaches and insights into the CYP3A7-CYP3A4 switch at young ages.

    Strougo, Ashley; Yassen, Ashraf; Monnereau, Claire; Danhof, Meindert; Freijer, Jan

    2014-09-01

    First-dose-in-children relies on the prediction of clearance from adults for which little information is available on the accuracy of the scaling-approaches applied. For CYP3A-metabolized compounds, scaling of clearance is further challenged by different isoforms and by the CYP3A7 to CYP3A4 switch at young ages. This investigation aimed to evaluate the accuracy of two frequently used scaling approaches and to gain insights into the ontogeny of CYP3A. Hence, a literature database was compiled containing 203 clearance values from term-neonates to adults for 18 CYP3A-metabolized compounds. The clearances in adults were scaled to children using (i) allometric scaling plus maturation function and (ii) a mechanistic approach based on the well-stirred model. Three maturation functions were separately evaluated. In children >3 months, all approaches were interchangeable heeding the maturation function applied and biases were mostly observed in children <3 months. The results from a sensitivity analysis indicate that these biases are possibly caused by disregarding the CYP3A7 activity which could account for up to 86% of the metabolism in term-neonates. Only the mechanistic approach using an overall-CYP3A maturation function led to unbiased predictions of clearances across all ages. The current investigation adds to the predictions of the first-dose-in-children of compounds (partially) metabolized by CYP3A. PMID:24676942

  15. Characterisation of Novel Cytochrome P450-fusion enzymes

    Luciakova, Dominika

    2015-01-01

    Characterisation of Novel Cytochrome P450-fusion enzymesSeptember 2014This study focuses on the characterisation of three novel cytochrome P450-partner (P450-fusion) enzymes of unknown structure and function. Despite several well-established P450 functions, new structures of P450s are published frequently, with the P450-redox partner fusion systems being among the most discussed, due to their enhanced activity and biotechnological potential. Other, more intriguing, P450-fusions involve partne...

  16. Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity

    Joshua Kellogg; Grace, Mary H.; Mary Ann Lila

    2014-01-01

    Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effec...

  17. Perspectives in developing industrial enzymes by using technological intelligence

    Óscar Fernando Castellanos Domínguez; Diana Cristina Ramírez Martínez; Víctor Mauricio Montañez

    2010-01-01

    Developing emergent technologies implies an ongoing challenge involving using new technological management tools. Enzyme engineering and its respective technology (within the context of biotechnology) is one of the areas of knowledge from which great expectations are constantly arising, such as providing competitive advantage in emergent economies like that of Colombia. However, several decades of research in this field have still not led to important results in terms of enzyme production an...

  18. Innovative enzymes for bioethanol production from lignocellulosic materials

    Marcolongo, Loredana

    2015-01-01

    The general aim of this work was to add new knowledge on novel hemicellulolytic enzymes involved in the hydrolysis of lignocellulosic materials, considered as a key process for the bioethanol production. Therefore, it is not only focused on (hemi)cellulolytic enzymes from mesophilic fungi and bacteria but also on newly isolated and characterized xylanase and β-xylosidase from the thermophilic bacteria Geobacillus thermodenitrificans A333 and Anoxybacillus sp. 3M, respectively. The cove...

  19. Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

    Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14C-hexose to 14C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

  20. Enzyme polymorphisms in Canarium

    Fifty-two accessions of Canarium involving seven species, C. ovatum, C. album, C. megalanthum, C. harveyi, C. indicum, C. mehenbethene, and C. odontophyllum were studied for isozyme polymorphisms. Starch gel electrophoresis with a histidine-citrate buffer system (pH 6.5) was employed to assay six en...

  1. Negative cooperativity in regulatory enzymes.

    Levitzki, A; Koshland, D E

    1969-04-01

    Negative cooperativity has been observed in CTP synthetase, an allosteric enzyme which contains a regulatory site. Thus, the same enzyme exhibits negative cooperativity for GTP (an effector) and glutamine (a substrate) and positive cooperativity for ATP and UTP (both substrates). In the process of the delineation of these phenomena, diagnostic procedures for negative cooperativity were developed. Application of these procedures to other enzymes indicates that negative cooperativity is a characteristic of many of them. These findings add strong support for the sequential model of subunit interactions which postulates that ligand-induced conformational changes are responsible for regulatory and cooperative phenomena in enzymes. PMID:5256410

  2. Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis.

    Arvind Ramanathan

    2011-11-01

    Full Text Available Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme-substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme-substrate interactions

  3. Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

    José Maria Rodrigues da Luz

    2012-12-01

    Full Text Available The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse. The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase and hydrolytic enzymes (cellulases, xylanases and tanases. Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6. These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

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

    Xianqin Wang

    2013-12-01

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

  5. Enzyme induction in cryopreserved human hepatocyte cultures.

    Kafert-Kasting, Sabine; Alexandrova, Krassimira; Barthold, Marc; Laube, Britta; Friedrich, Gerhard; Arseniev, Lubomir; Hengstler, Jan G

    2006-03-15

    Freshly isolated human hepatocytes are considered as the gold standard for in vitro testing of drug candidates. Meanwhile also cryopreserved human hepatocyte suspensions are available. However, a drawback of these cells is the incalculability of attachment to the culture dish. Therefore, we established a technique freezing hepatocytes cultured on a collagen gel. After thawing damaged cells were removed to a certain extent by gentle washing with culture medium prior to adding an upper gel layer. The morphology of the resulting hepatocyte cultures could not be distinguished from that of non-frozen cells. However, basal activities of cytochrome P450 isoforms decreased in cryopreserved compared to non-frozen hepatocytes, as evidenced by analysis of testosterone hydroxylation (OHT) in positions 6beta, 16alpha, 2beta and 6alpha. Nevertheless, enzyme induction factors caused by 24 h incubation with 50 microM rifampicin were similar in cryopreserved and non-frozen hepatocytes. In cryopreserved hepatocytes rifampicin caused an increase in mean values of 6beta-OHT formation from 57.2 to 157.7 pmol/well/min (2.8-fold), compared to an increase from 115.8 to 269.1 pmol/well/min (2.3-fold) in non-frozen cells. Similarly, 16alpha- and 2beta-OHT showed induction factors of 2.4- and 2.3-fold in cryopreserved compared to 1.6- and 2.4-fold in non-frozen hepatocytes, respectively. In conclusion, human hepatocytes cryopreserved on collagen gels show a clear induction of CYP3A4 by rifampicin, although the basal activities are reduced compared to non-frozen cells. PMID:16473453

  6. Inhibitory effect of mitragynine on human cytochrome P450 enzyme activities

    N A Hanapi

    2013-01-01

    Full Text Available Context: To date, many findings reveal that most of the modern drugs have the ability to interact with herbal drugs. Aims: This study was conducted to determine the inhibitory effects of mitragynine on cytochrome P450 2C9, 2D6 and 3A4 activities. Methods and Material: The in vitro study was conducted using a high-throughput luminescence assay. Statistical Analysis: Statistical analysis was conducted using one-way ANOVA and Dunnett′s test with P < 0.05 vs. control. The IC 50 values were calculated using the GraphPad Prism® 5 (Version 5.01, GraphPad Software, Inc., USA. Results: Assessment using recombinant enzymes showed that mitragynine gave the strongest inhibitory effect on CYP2D6 with an IC 50 value of 0.45±0.33 mM, followed by CYP2C9 and CYP3A4 with IC 50 values of 9.70±4.80 and 41.32±6.74 mM respectively. Positive inhibitors appropriate for CYP2C9, CYP2D6, and CYP3A4 which are sulfaphenazole, quinidine and ketoconazole were used respectively. V max values of CYP2C9, CYP2D6 and CYP3A4 were 0.0005, 0.01155 and 0.0137 mM luciferin formed/pmol/min respectively. K m values of CYP2C9, CYP2D6, and CYP3A4 were 32.65, 56.01, and 103.30 mM respectively. Mitragynine noncompetitively inhibits CYP2C9 and CYP2D6 activities with the K i values of 61.48 and 12.86 mM respectively. On the other hand, mitragynine inhibits CYP3A4 competitively with a K i value of 379.18 mM. Conclusions: The findings of this study reveal that mitragynine might inhibit cytochrome P450 enzyme activities, specifically CYP2D6. Therefore, administration of mitragynine together with herbal or modern drugs which follow the same metabolic pathway may contribute to herb-drug interactions.

  7. Phage lytic enzymes: a history

    David; Trudil

    2015-01-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters’ or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well(Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay(Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes–from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  8. Enzymic hydrolysis of chlorella cells

    Khraptsova, G.I.; Tsaplina, I.A.; Burdenko, L.G.; Khoreva, S.L.; Loginova, L.G.

    1981-01-01

    Treatment of C. ellipsoidea, C. pyrenoidosa, and C. vulgaris with cellulolytic enzymes (from Aspergillus terreus) and pectofoetidin p10x (from A. foetidus) resulted in the degradation and lysis of the algae cells. The cells were more sensitive to cellulase than to pectinase. The combination of both enzymes produced a synergistic effect on cell lysis.

  9. Enzyme catalysis: Evolution made easy

    Wee, Eugene J. H.; Trau, Matt

    2014-09-01

    Directed evolution is a powerful tool for the development of improved enzyme catalysts. Now, a method that enables an enzyme, its encoding DNA and a fluorescent reaction product to be encapsulated in a gel bead enables the application of directed evolution in an ultra-high-throughput format.

  10. Enzyme immunoassay for human ferritin

    We described an enzyme immunoassay with use of β-D-galactosidase for quantitation of ferritin in human serum. The minimum detectable ferritin concentration is 0.25 μg/L of serum, which is comparable to results obtained by radioimmunoassay. The correlation coefficient between values determined by enzyme immunoassay and radioimmunoassay was 0.95

  11. Radiation inactivation of proteolytic enzymes

    The survey was devoted to generalization of protease inactivation mechanism for different conditions of irradiation and for different kinds of enzymes. The importance of radiation conformation changes and the possible use of radiolytic processes were considered especially. The serine-, SH-, acidic-and metal-contained enzymes were described

  12. Selective inhibition of human cytochrome P450 3A4 by N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-(1, 1-dimethylethylaminocarbonyl)-4-[(furo[2, 3-b]pyridin-5-yl)methyl]piperazin-1-yl]-4(S)-hydroxy-2(R)-phenylmethy lpentanamide and P-glycoprotein by valspodar in gene transfectant systems.

    Kawahara, I; Kato, Y; Suzuki, H; Achira, M; Ito, K; Crespi, C L; Sugiyama, Y

    2000-10-01

    Our previous report showed that L754.394 and valspodar (PSC833) are potent inhibitors of midazolam hydroxylation in human jejunum microsomes and vectorial transport of vinblastine in Caco-2 cells, respectively. In the present study, to directly examine the interactions of these compounds as well as other substrates with CYP3A4 and P-glycoprotein (P-gp), we performed in vitro inhibition studies using recombinant CYP3A4-expressed microsomes and an MDR1-transfected cell line, LLC-MDR1, respectively. In CYP3A4-expressed microsomes, both L754.394 and ketoconazole, at a concentration less than 0.5 microM, are the most potent inhibitors of the formation of 1'-hydroxymidazolam, a major metabolite of midazolam formed by CYP3A4. The greatest inhibitory effect on the transcellular transport of digoxin in LLC-MDR1 cells was observed in the presence of valspodar (<0.1 microM), followed by verapamil. From a comparison of the IC(50) values, it was shown that L754.394 and valspodar exhibited the highest selectivity for CYP3A4 and P-gp, respectively. To demonstrate such specificity, both midazolam hydroxylation and digoxin transport were observed in CYP3A4 transfected Caco-2 cells, which coexpress both P-gp and CYP3A4, in the presence or absence of L754.394 (0.5 microM) and valspodar (1.0 microM). L754.394 almost completely inhibited midazolam hydroxylation, but not digoxin transport, whereas almost complete inhibition of digoxin transport was observed in the presence of valspodar, but inhibition of the hydroxylation was minimal. Thus, the present study has demonstrated that L754.394 has a specific inhibitory effect on CYP3A4, whereas valspodar is specific for P-gp. PMID:10997946

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

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

    2015-12-01

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

  14. Frequency of anti-glycoprotein Ia/IIa (anti-HPA-5b,-5a and anti-glycoprotein IIb/IIIa (anti-HPA-1a,-3a,-4a alloantibodies in multiparous women of African descent

    Zaccheaus A Jeremiah

    2010-05-01

    Full Text Available Zaccheaus A Jeremiah1, Justina E Oburu2, Osaro Erhabor1, Fiekumo I Buseri1, Teddy C Adias31Haematology and Blood Transfusion Science Unit, Department of Medical Laboratory Sciences, College of Health Sciences, Niger Delta University, Wilberforce Island, Nigeria; 2Department of Haematology and Blood Transfusion, University of Port Harcourt Teaching Hospital, Port Harcourt, Nigeria; 3Rivers State University of Science and Technology, Port Harcourt, NigeriaBackground: Human platelet antibodies are often implicated in some disease conditions, such as neonatal alloimmune thrombocytopenia (NAIT, idiopathic thrombocytopenic purpura (ITP and platelet refractoriness. The frequencies of these alloantibodies have not been reported in Nigeria and West Africa.Methods: Screening for allontibodies to human platelet antigens (HPA was undertaken using the GTI PakPlus® qualitative solid phase ELISA reagent. Platelet count was done using the ICSH approved procedure using 1% ammonium oxalate reagent.Study design: A cross-section of apparently healthy adult Nigerian multiparous non-pregnant women, who were staff of a tertiary health facility in the Niger Delta, Nigeria, were screened for alloantibodies to human platelet antigens.Results: Of the one hundred (100 women screened, the prevalence of anti-glycoprotein IIb/IIIa (anti-HPA-Ia,-3a,-4a was zero percent (0%, anti-glycoprotein Ia/IIa (anti-HPA-5b accounted for 30% of results, while anti-glycoprotein Ia/IIa (anti-HPA-5a was 18%. Parity was found to exert significant influence on the development to HPA antibodies (Fisher’s Exact Test = 11.683, P < 0.05; 13.577, P < 0.01. The platelet count of the women did not appear to exert any influence on the development of the antibodies (P > 0.05.Conclusion: This study has observed a high prevalence of anti-HPA-5b in our sample population. The prevalence of alloantibodies to HPA antigens was found to associate strongly with parity. These results indicate that there is a

  15. Human bone marrow niche chemoprotection mediated by cytochrome P450 enzymes.

    Alonso, Salvador; Su, Meng; Jones, Jace W; Ganguly, Sudipto; Kane, Maureen A; Jones, Richard J; Ghiaur, Gabriel

    2015-06-20

    Substantial evidence now demonstrates that interactions between the tumor microenvironment and malignant cells are a critical component of clinical drug resistance. However, the mechanisms responsible for microenvironment-mediated chemoprotection remain unclear. We showed that bone marrow (BM) stromal cytochrome P450 (CYP)26 enzymes protect normal hematopoietic stem cells (HSCs) from the pro-differentiation effects of retinoic acid. Here, we investigated if stromal expression of CYPs is a general mechanism of chemoprotection. We found that similar to human hepatocytes, human BM-derived stromal cells expressed a variety of drug-metabolizing enzymes. CYP3A4, the liver's major drug-metabolizing enzyme, was at least partially responsible for BM stroma's ability to protect multiple myeloma (MM) and leukemia cells from bortezomib and etoposide, respectively, both in vitro and in vivo. Moreover, clarithromycin overcame stromal-mediated MM resistance to dexamethasone, suggesting that CYP3A4 inhibition plays a role in its ability to augment the activity of lenalidomide and dexamethasone as part of the BiRd regimen. We uncovered a novel mechanism of microenvironment-mediated drug resistance, whereby the BM niche creates a sanctuary site from drugs. Targeting these sanctuaries holds promise for eliminating minimal residual tumor and improving cancer outcomes. PMID:25915157

  16. Hfq stimulates the activity of the CCA-adding enzyme

    Betat Heike

    2007-10-01

    Full Text Available Abstract Background The bacterial Sm-like protein Hfq is known as an important regulator involved in many reactions of RNA metabolism. A prominent function of Hfq is the stimulation of RNA polyadenylation catalyzed by E. coli poly(A polymerase I (PAP. As a member of the nucleotidyltransferase superfamily, this enzyme shares a high sequence similarity with an other representative of this family, the tRNA nucleotidyltransferase that synthesizes the 3'-terminal sequence C-C-A to all tRNAs (CCA-adding enzyme. Therefore, it was assumed that Hfq might not only influence the poly(A polymerase in its specific activity, but also other, similar enzymes like the CCA-adding enzyme. Results Based on the close evolutionary relation of these two nucleotidyltransferases, it was tested whether Hfq is a specific modulator acting exclusively on PAP or whether it also influences the activity of the CCA-adding enzyme. The obtained data indicate that the reaction catalyzed by this enzyme is substantially accelerated in the presence of Hfq. Furthermore, Hfq binds specifically to tRNA transcripts, which seems to be the prerequisite for the observed effect on CCA-addition. Conclusion The increase of the CCA-addition in the presence of Hfq suggests that this protein acts as a stimulating factor not only for PAP, but also for the CCA-adding enzyme. In both cases, Hfq interacts with RNA substrates, while a direct binding to the corresponding enzymes was not demonstrated up to now (although experimental data indicate a possible interaction of PAP and Hfq. So far, the basic principle of these stimulatory effects is not clear yet. In case of the CCA-adding enzyme, however, the presented data indicate that the complex between Hfq and tRNA substrate might enhance the product release from the enzyme.

  17. Database of ligand-induced domain movements in enzymes

    Hayward Steven

    2009-03-01

    Full Text Available Abstract Background Conformational change induced by the binding of a substrate or coenzyme is a poorly understood stage in the process of enzyme catalysed reactions. For enzymes that exhibit a domain movement, the conformational change can be clearly characterized and therefore the opportunity exists to gain an understanding of the mechanisms involved. The development of the non-redundant database of protein domain movements contains examples of ligand-induced domain movements in enzymes, but this valuable data has remained unexploited. Description The domain movements in the non-redundant database of protein domain movements are those found by applying the DynDom program to pairs of crystallographic structures contained in Protein Data Bank files. For each pair of structures cross-checking ligands in their Protein Data Bank files with the KEGG-LIGAND database and using methods that search for ligands that contact the enzyme in one conformation but not the other, the non-redundant database of protein domain movements was refined down to a set of 203 enzymes where a domain movement is apparently triggered by the binding of a functional ligand. For these cases, ligand binding information, including hydrogen bonds and salt-bridges between the ligand and specific residues on the enzyme is presented in the context of dynamical information such as the regions that form the dynamic domains, the hinge bending residues, and the hinge axes. Conclusion The presentation at a single website of data on interactions between a ligand and specific residues on the enzyme alongside data on the movement that these interactions induce, should lead to new insights into the mechanisms of these enzymes in particular, and help in trying to understand the general process of ligand-induced domain closure in enzymes. The website can be found at: http://www.cmp.uea.ac.uk/dyndom/enzymeList.do

  18. Applications of Nanomaterials in Electrochemical Enzyme Biosensors

    Xiaodi Yang

    2009-10-01

    Full Text Available A biosensor is defined as a kind of analytical device incorporating a biological material, a biologically derived material or a biomimic intimately associated with or integrated within a physicochemical transducer or transducing microsystem. Electrochemical biosensors incorporating enzymes with nanomaterials, which combine the recognition and catalytic properties of enzymes with the electronic properties of various nanomaterials, are new materials with synergistic properties originating from the components of the hybrid composites. Therefore, these systems have excellent prospects for interfacing biological recognition events through electronic signal transduction so as to design a new generation of bioelectronic devices with high sensitivity and stability. In this review, we describe approaches that involve nanomaterials in direct electrochemistry of redox proteins, especially our work on biosensor design immobilizing glucose oxidase (GOD, horseradish peroxidase (HRP, cytochrome P450 (CYP2B6, hemoglobin (Hb, glutamate dehydrogenase (GDH and lactate dehydrogenase (LDH. The topics of the present review are the different functions of nanomaterials based on modification of electrode materials, as well as applications of electrochemical enzyme biosensors.

  19. Radiofluorinated enzyme probes of dopaminergic function

    6-[18F]Fluoro-L-DOPA (FDOPA), a functional analog of exogenous L-DOPA is useful to determine in-vivo aromatic amino acid decarboxylase (AAAD) mediated decarboxylation and apparent dopamine turnover rates. Also, 4- (4-FMT) and 6-[18F]fluoro-L-m-tyrosines (6-FMT) are analogs that essentially follow the L-DOPA pathway of central metabolism, without 3-O methylation or extensive peripheral metabolism. Interestingly, molecular and enzymatic mechanisms involved in their retention in central brain structures differ substantially. In addition, radiofluorinated enzyme probes based on the principle of dual-enzyme (AAAD-MAO) inhibition are discussed from the standpoint of stereo-and geometric isomerisms on the enzymatic behavior and central kinetics in primates. Finally, analogs that bind selectively and covalently to AAAD (e.g. α-fluoromethyl-6-[18F]fluoro-L-DOPA) are introduced. All these analogs are presented in a context of a useful strategy for designing radiotracers to probe specific enzymes and their functions

  20. School Parent Involvement Policy

    Center for Law and Education (NJ3), 2005

    2005-01-01

    This school parent involvement policy is divided into three sections: (1) Development and Adoption of the Parent Involvement Policy; (2) Contents of the Parent Involvement Policy; and (3) Distributing and Revising the School's Parent Involvement Policy. This paper presents the provision of the Section 1118 of Title I of the No Child Left Behind…

  1. BAKERY ENZYMES IN CEREAL TECHNOLOGIES

    Václav Koman

    2012-10-01

    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE Bread is the most common and traditional food in the world. For years, enzymes such as malt and fungal alpha-amylase have been used in bread making. Due to the changes in the baking industry and the ever-increasing demand for more natural products, enzymes have gained real importance in bread-making. If an enzyme is added, it is often destroyed by the heat during the baking process. For generations, enzymes have been used for the improvement of texture and appearance, enhancement of nutritional values and generation of appealing flavours and aromas. Enzymes used in bakery industry constitute nearly one third of the market. The bakery products have undergone radical improvements in quality over the past years in terms of flavour, texture and shelf-life. The the biggest contributor for these improvementsis the usage of enzymes. Present work seeks to systematically describe bakery enzymes, their classification, benefits, usage and chemical reactions in the bread making process.doi:10.5219/193

  2. HIV-1 Alters Intestinal Expression of Drug Transporters and Metabolic Enzymes: Implications for Antiretroviral Drug Disposition.

    Kis, Olena; Sankaran-Walters, Sumathi; Hoque, M Tozammel; Walmsley, Sharon L; Dandekar, Satya; Bendayan, Reina

    2016-05-01

    This study investigated the effects of HIV-1 infection and antiretroviral therapy (ART) on the expression of intestinal drug efflux transporters, i.e., P-glycoprotein (Pgp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP), and metabolic enzymes, such as cytochrome P450s (CYPs), in the human upper intestinal tract. Intestinal biopsy specimens were obtained from HIV-negative healthy volunteers, ART-naive HIV-positive (HIV(+)) subjects, and HIV(+) subjects receiving ART (10 in each group). Intestinal tissue expression of drug transporters and metabolic enzymes was examined by microarray, real-time quantitative reverse transcription-PCR (qPCR), and immunohistochemistry analyses. Microarray analysis demonstrated significantly lower expression of CYP3A4 and ABCC2/MRP2 in the HIV(+) ART-naive group than in uninfected subjects. qPCR analysis confirmed significantly lower expression of ABCC2/MRP2 in ART-naive subjects than in the control group, while CYP3A4 and ABCG2/BCRP showed a trend toward decreased expression. Protein expression of MRP2 and BCRP was also significantly lower in the HIV(+) naive group than in the control group and was partially restored to baseline levels in HIV(+) subjects receiving ART. In contrast, gene and protein expression of ABCB1/Pgp was significantly increased in HIV(+) subjects on ART relative to HIV(+) ART-naive subjects. These data demonstrate that the expression of drug-metabolizing enzymes and efflux transporters is significantly altered in therapy-naive HIV(+) subjects and in those receiving ART. Since CYP3A4, Pgp, MRPs, and BCRP metabolize or transport many antiretroviral drugs, their altered expression with HIV infection may negatively impact drug pharmacokinetics in HIV(+) subjects. This has clinical implications when using data from healthy volunteers to guide ART. PMID:26902756

  3. In Vitro Effects of Concomitant Use of Herbal Preparations on Cytochrome P450s Involved in Clozapine Metabolism.

    Wang, Wei; Tian, Dan-Dan; Zhang, Zhang-Jin

    2016-01-01

    Herbal supplements are increasingly used in psychiatric practice. Our epidemiological study has identified several herbal preparations associated with adverse outcomes of antipsychotic therapy. In this study, we evaluated the in vitro effects of four herbal preparations-Radix Rehmanniae (RR), Fructus Schisandrae (FS), Radix Bupleuri (RB) and Fructus Gardeniae (FG)-on cytochrome P450s (CYPs) involved in the metabolism of clozapine in human liver microsomes (HLMs) and recombinant human cytochrome P450 enzymes (rCYPs). N-desmethylclozapine and clozapine N-oxide, two major metabolites of clozapine, were measured using high-performance liquid chromatography (HPLC). FG, RR and RB showed negligible inhibitory effects in both in vitro systems, with estimated half-maximal inhibitory concentrations (IC50) and apparent inhibitory constant values (Ki) greater than 1 mg/mL (raw material), suggesting that minimal metabolic interaction occurs when these preparations are used concomitantly with clozapine. The FS extract affected CYP activity with varying potency; its effect on CYP 3A4-catalyzed clozapine oxidation was relatively strong (Ki: 0.11 mg/mL). Overall, the weak-to-moderate inhibitory effect of FS on in vitro clozapine metabolism indicated its potential role in herb-drug interaction in practice. PMID:27164071

  4. The surface science of enzymes

    Rod, Thomas Holm; Nørskov, Jens Kehlet

    One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function? To...... exist and the two kinds of catalyst can be described by similar tools, nature and human effort have come up with different solutions. This on the other hand implies that new and improved catalysts may be made by learning from nature....

  5. An enzyme with rhamnogalacturonase activity.

    Kovod, L.V.; Dalboge, H; Andersen, L N; Kauppinen, M.; Christgan, S.; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A. G. J.; Schols, H.A.

    1994-01-01

    An enzyme exhibiting rhamnogalacturonase activity, which enzyme: a) is encoded by the DNA sequence shown in SEQ ID No. 1 or a sequence homologous thereto encoding a polypeptide with RGase activity, b) has the amino acid sequence shown in SEQ ID No. 2 or an analogue thereof, c) is reactive with an antibody raised against the enzyme encoded by the DNA sequence shown in SEQ ID No. 1, d) has a pH optimum above pH 5, and/or e) has a relative activity of at least 30t a pH in the range of 5.5-6.5. T...

  6. Detoxification of azo dyes by bacterial oxidoreductase enzymes.

    Mahmood, Shahid; Khalid, Azeem; Arshad, Muhammad; Mahmood, Tariq; Crowley, David E

    2016-08-01

    Azo dyes and their intermediate degradation products are common contaminants of soil and groundwater in developing countries where textile and leather dye products are produced. The toxicity of azo dyes is primarily associated with their molecular structure, substitution groups and reactivity. To avoid contamination of natural resources and to minimize risk to human health, this wastewater requires treatment in an environmentally safe manner. This manuscript critically reviews biological treatment systems and the role of bacterial reductive and oxidative enzymes/processes in the bioremediation of dye-polluted wastewaters. Many studies have shown that a variety of culturable bacteria have efficient enzymatic systems that can carry out complete mineralization of dye chemicals and their metabolites (aromatic compounds) over a wide range of environmental conditions. Complete mineralization of azo dyes generally involves a two-step process requiring initial anaerobic treatment for decolorization, followed by an oxidative process that results in degradation of the toxic intermediates that are formed during the first step. Molecular studies have revealed that the first reductive process can be carried out by two classes of enzymes involving flavin-dependent and flavin-free azoreductases under anaerobic or low oxygen conditions. The second step that is carried out by oxidative enzymes that primarily involves broad specificity peroxidases, laccases and tyrosinases. This review focuses, in particular, on the characterization of these enzymes with respect to their enzyme kinetics and the environmental conditions that are necessary for bioreactor systems to treat azo dyes contained in wastewater. PMID:25665634

  7. Amperometric ATP biosensor based on polymer entrapped enzymes.

    Kueng, Angelika; Kranz, Christine; Mizaikoff, Boris

    2004-05-15

    A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1). PMID:15046763

  8. Printable enzyme-embedded materials for methane to methanol conversion.

    Blanchette, Craig D; Knipe, Jennifer M; Stolaroff, Joshuah K; DeOtte, Joshua R; Oakdale, James S; Maiti, Amitesh; Lenhardt, Jeremy M; Sirajuddin, Sarah; Rosenzweig, Amy C; Baker, Sarah E

    2016-01-01

    An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scale structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas-liquid reactions. PMID:27301270

  9. Emerging roles of deubiquitinating enzymes in human cancer1

    Jin-ming YANG

    2007-01-01

    Protein modifications by the covalent linkage of ubiquitin have significant in-volvement in many cellular processes, including stress response, oncogenesis,viral infection, transcription, protein turnover, organelle biogenesis, DNA repair,cellular differentiation, and cell cycle control. Protein ubiquitination and subse-quent degradation by the proteasome require the participation of both ubiquitinating enzymes and deubiquitinating enzymes. Although deubiquitinatingenzymes constitute a large family in the ubiquitin system, the study of this class of proteins is still in its infant stage. Recent studies have revealed a variety of molecular and biological functions of deubiquitinating enzymes and their associa-tion with human diseases. In this review we will discuss the possible roles that deubiquitinating enzymes may play in cancers.

  10. 2,6-Bis(2-hydroxyethyl-8b,8c-diphenylperhydro-2,3a,4a,6,7a,8a-hexaazacyclopenta[def]fluorene-4,8-dithione

    Zihua Wang

    2009-06-01

    Full Text Available In the title molecule, C24H28N6O2S2, the dihedral angle between the aromatic ring planes is 42.2 (1°. In the crystal structure, the hydroxy groups are involved in O—H...S hydrogen bonding, which links the molecules into corrugated layers propagating parallel to the bc plane.

  11. Pharmacogenomics of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and MDR1 in Vietnam

    2008-01-01

    Abstract Aim The aim of this study was to obtain pharmacogenetic data in a Vietnamese population on genes coding for proteins involved in the elimination of drugs currently used for the treatment of malaria and human immunodeficiency virus/acquired immunodeficiency syndrome. Method The main polymorphisms on the c...

  12. El juego con materiales manipulativos para mejorar el aprendizaje de las matemáticas en Educación Infantil: Una propuesta para niños y niñas de 3 a 4 años

    Marta Berga Espona

    2013-12-01

    Full Text Available Elaboración de una propuesta para niños de 3 a 4 años de edad, llevada a la práctica, y orientada al aprendizaje de las matemáticas, en la que se da importancia al juego y a la manipulación de objetos, a partir del juego de construcciones con cuerpos geométricos. La propuesta consta de nueve actividades. Partiendo de unos objetivos adecuados para estas edades, proponemos actividades de juego libre, otras más dirigidas, y otras que implican representaciones mentales más avanzadas. Se tiene en cuenta el alumnado, sus características, sus habilidades y su ritmo de desarrollo. Los niños y niñas tienen un papel activo y son protagonistas de su propio aprendizaje, para tratar de conseguir un aprendizaje significativo. Play with manipulatives to improve the learning of mathematics in the early childhood: A proposal for children from 3 to 4 years. This work project involves the development of a proposal for children from 3 to 4 years old, taking it to practice for a mathematical learning and giving importance to manipulate objects from block play. This proposal is based in play, and is composed by nine activities, with are developmentally appropriate for these ages. There will be free play activities, others will be more directed and others must make mental representations. The activities proposed take into account the students, their characteristics, their skills and their pace of development. All the students will have an active role and will be responsible for their own learning, trying to achieve a meaningful learning. Keywords: Childhood Education, meaningful learning, mathematics, manipulatives, play, creativity.

  13. ORGANOPHOSPHATE DEGRADING ENZYMES - PHASE I

    Agave BioSystems in collaboration with Carl A. Batt proposes to develop decon-nanoparticles, which will leverage ongoing opportunities in enzyme engineering and the fabrication of functionalized magnetic nanoparticles. Enhanced performance will be engineered into the system t...

  14. Controlled enzyme catalyzed heteropolysaccharide degradation

    Rasmussen, Louise Enggaard

    The work presented in this PhD thesis has provided a better understanding of the enzyme kinetics and quantitative phenomena of the hydrolysis of xylan substrates by selected pure enzyme preparations. Furthermore, the options for producing specific substituted xylooligosaccharides from selected...... substrates by specific xylanase treatment have been examined. The kinetics of the enzymatic degradation of water-extractable wheat arabinoxylan (WE-AX) during designed treatments with selected monocomponent enzymes was investigated by monitoring the release of xylose and arabinose. The results of different...... effects between -xylosidase and the α-L-arabinofuranosidases on the xylose release were low as compared to the effect of xylanase addition with β-xylosidase, which increased the xylose release by ~25 times in 30 minutes. At equimolar addition levels of the four enzymes, the xylanase activity was thus rate...

  15. Enzyme Teaching by a Virtual Laboratory

    J.K. Sugai

    2010-05-01

    Full Text Available Biochemistry learning demands skills to obtaining and interpreting the experimental data. In a classical model of teaching involve student’s hands-on participation. However this model is expensive, not safe and should be carried out in a short and limited time course. With utilization of educational software these disadvantages are overcome, since the virtual activity could be realized at free full access, and is a tool for individual study. The aim of the present work is to present educational software focused on a virtual for undergraduate student of biochemistry courses. The software development was performed with the help of concept maps, ISIS Draw, ADOBE Photoshop and FLASH MX Program applied on the subject salivary amylase. It was possible to present the basic methodologies for study of the kinetic of enzyme. The substrate (starch consumption was determinate by iodine reaction, while the products (reducing sugars formation was evaluated by cupper-alkaline reaction. The protocols of the virtual experiments are present verbally as well as a subtitle. A set of exercises are disposable, which allowed an auto evaluation and a review of the subject. The experimental treatment involved the presentation of this hypermedia for Nutrition and Dentistry/UFSC undergraduate students as a tool for better comprehension of the theme and promoted the understanding of the kinetic of enzyme.

  16. A microarray-based system for the simultaneous analysis of single nucleotide polymorphisms in human genes involved in the metabolism of anti-malarial drugs

    Qi Weihong

    2009-12-01

    Full Text Available Abstract Background In order to provide a cost-effective tool to analyse pharmacogenetic markers in malaria treatment, DNA microarray technology was compared with sequencing of polymerase chain reaction (PCR fragments to detect single nucleotide polymorphisms (SNPs in a larger number of samples. Methods The microarray was developed to affordably generate SNP data of genes encoding the human cytochrome P450 enzyme family (CYP and N-acetyltransferase-2 (NAT2 involved in anti-malarial drug metabolisms and with known polymorphisms, i.e. CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, and NAT2. Results For some SNPs, i.e. CYP2A6*2, CYP2B6*5, CYP2C8*3, CYP2C9*3/*5, CYP2C19*3, CYP2D6*4 and NAT2*6/*7/*14, agreement between both techniques ranged from substantial to almost perfect (kappa index between 0.61 and 1.00, whilst for other SNPs a large variability from slight to substantial agreement (kappa index between 0.39 and 1.00 was found, e.g. CYP2D6*17 (2850C>T, CYP3A4*1B and CYP3A5*3. Conclusion The major limit of the microarray technology for this purpose was lack of robustness and with a large number of missing data or with incorrect specificity.

  17. Enzyme Assay: An Investigative Approach to Enhance Science Process Skills

    Vartak, Rekha; Ronad, Anupama; Ghanekar, Vikrant

    2013-01-01

    Scientific investigations play a vital role in teaching and learning the process of science. An investigative task that was developed for pre-university students is described here. The task involves extraction of an enzyme from a vegetable source and its detection by biochemical method. At the beginning of the experiment, a hypothesis is presented…

  18. Engineering Cellulase Enzymes for Bioenergy

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  19. [The rise of enzyme engineering in China].

    Li, Gaoxiang

    2015-06-01

    Enzyme engineering is an important part of the modern biotechnology. Industrial biocatalysis is considered the third wave of biotechnology following pharmaceutical and agricultural waves. In 25 years, China has made a mighty advances in enzyme engineering research. This review focuses on enzyme genomics, enzyme proteomics, biosynthesis, microbial conversion and biosensors in the Chinese enzyme engineering symposiums and advances in enzyme preparation industry in China. PMID:26672358

  20. Methyl 1-methyl-3-p-tolyl-1,2,3,3a,4,11c-hexahydrobenzo[f]chromeno[4,3-b]pyrrole-3a-carboxylate

    S. Nirmala

    2009-08-01

    Full Text Available In the title compound, C25H25NO3, the dihydropyran ring adopts a half-chair conformation, whereas the pyrrolidine ring is in a twist conformation. The tolyl group is oriented at an angle of 82.92 (7° with respect to the napthalene ring system. In the crystal structure, molecules are linked into centrosymmetric dimers by C—H...π interactions involving the benzene ring of the tolyl group.

  1. 25. Steenbock symposium -- Biosynthesis and function of metal clusters for enzymes: Proceedings

    NONE

    1997-12-31

    This symposium was held June 10--14, 1997 in Madison, Wisconsin. The purpose of this conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on biochemistry of enzymes that have an affinity for metal clusters. Attention is focused on the following: metal clusters involved in energy conservation and remediation; tungsten, molybdenum, and cobalt-containing enzymes; Fe proteins, and Mo-binding proteins; nickel enzymes; and nitrogenase.

  2. Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials.

    Bronnenmeier, K; Kern, A.; Liebl, W.; Staudenbauer, W L

    1995-01-01

    A separation procedure for the analysis of the enzyme components of the hyperthermophilic bacterium Thermotoga maritima involved in cellulose and xylan degradation was developed. Resolution of the enzymes was achieved by a combination of fast protein liquid chromatography anion exchange and hydrophobic interaction chromatography. Enzyme fractions were assayed for hydrolysis of Avicel, carboxymethylcellulose (CMC), beta-glucan, laminarin, xylan, p-nitrophenyl-beta-D-glucoside, p-nitrophenyl-be...

  3. The synthesis of starch from carbon dioxide using isolubilized stabilized enzymes

    Bassham, J. A.; Bearden, L.; Wilke, C.; Carroad, P.; Mitra, G.; Ige, R.

    1972-01-01

    Systems for artificial manufacture of starch and for delineation of technological areas, and the rationale for studying them are considered. A discussion of the enzyme-catalyzed routes of synthesis available and a choice as to the most promising route are presented. A discussion of the enzymes involved, of enzyme insolubilization technology, and of possible engineering approaches, with examples in the form of model calculations for both reactors and separators, are also presented.

  4. Insights on Cytochrome P450 Enzymes and Inhibitors Obtained Through QSAR Studies

    Maryam Foroozesh

    2012-08-01

    Full Text Available The cytochrome P450 (CYP superfamily of heme enzymes play an important role in the metabolism of a large number of endogenous and exogenous compounds, including most of the drugs currently on the market. Inhibitors of CYP enzymes have important roles in the treatment of several disease conditions such as numerous cancers and fungal infections in addition to their critical role in drug-drug interactions. Structure activity relationships (SAR, and three-dimensional quantitative structure activity relationships (3D-QSAR represent important tools in understanding the interactions of the inhibitors with the active sites of the CYP enzymes. A comprehensive account of the QSAR studies on the major human CYPs 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and a few other CYPs are detailed in this review which will provide us with an insight into the individual/common characteristics of the active sites of these enzymes and the enzyme-inhibitor interactions.

  5. New protein involved in the replacement of cell molecules

    Poulsen, Jesper Buchhave

    2011-01-01

    In collaboration with colleagues from La Trobe University, Australia, scientists at Aarhus University have discovered and defined a novel enzyme involved in the replacement and renewal of cell molecules. The enzyme exerts its function within the so-called mitochondria - small “enclosed” compartme......” compartments in the human cell - where most cellular energy is produced. The findings may ultimately explain the cause of certain diseases with relation to the mitochondria.......In collaboration with colleagues from La Trobe University, Australia, scientists at Aarhus University have discovered and defined a novel enzyme involved in the replacement and renewal of cell molecules. The enzyme exerts its function within the so-called mitochondria - small “enclosed...

  6. Characterization of three amidinotransferases involved in the biosynthesis of ketomemicins.

    Ogasawara, Yasushi; Fujimori, Michiko; Kawata, Junpei; Dairi, Tohru

    2016-08-01

    We recently reported a novel class of amide bond forming enzymes (peptide ligases) involved in the biosynthesis of pheganomycins, resorcinomycins and ketomemicins. This class of enzymes exclusively utilizes Nα-amidino amino acids as the N-terminal substrate. In this Letter, we characterized three new amidinotransferases involved in the biosynthesis of ketomemicins and showed that l-arginine was the amidino-acceptor of amidinotransferases in both the Micromonospora sp. and Streptomyces mobaraensis clusters, while the Salinispora tropica enzyme recognized l-valine. Unexpectedly, the S. tropica enzyme accepted several different amino acids as amidino acceptors in addition to l-valine. Accordingly, we re-investigated the specific metabolites governed by the gene cluster of S. tropica and identified several minor congeners of ketomemicin C with different N-terminal amidino-amino acids. These results indicate that the amidinotransferase of S. tropica is promiscuous and could be useful to generate new ketomemicin-type natural products. PMID:27289319

  7. The Kinetics of Enzyme Mixtures

    Simon Brown

    2014-03-01

    Full Text Available Even purified enzyme preparations are often heterogeneous. For example, preparations of aspartate aminotransferase or cytochrome oxidase can consist of several different forms of the enzyme. For this reason we consider how different the kinetics of the reactions catalysed by a mixture of forms of an enzyme must be to provide some indication of the characteristics of the species present. Based on the standard Michaelis-Menten model, we show that if the Michaelis constants (Km of two isoforms differ by a factor of at least 20 the steady-state kinetics can be used to characterise the mixture. However, even if heterogeneity is reflected in the kinetic data, the proportions of the different forms of the enzyme cannot be estimated from the kinetic data alone. Consequently, the heterogeneity of enzyme preparations is rarely reflected in measurements of their steady-state kinetics unless the species present have significantly different kinetic properties. This has two implications: (1 it is difficult, but not impossible, to detect molecular heterogeneity using kinetic data and (2 even when it is possible, a considerable quantity of high quality data is required.

  8. Effects of genetic polymorphism of cytochrome P450 enzymes on the pharmacokinetics of benzodiazepines.

    Fukasawa, T; Suzuki, A; Otani, K

    2007-08-01

    Pharmacogenetic studies have shown that several cytochrome P450 (CYP) enzymes exhibit genetic polymorphisms. Several benzodiazepines (BZPs) are metabolized predominantly or partly by polymorphic CYP2C19 and CYP3A4/5. The pharmacokinetics of diazepam, etizolam, quazepam and desmethylclobazam have been shown to be affected by CYP2C19 polymorphism. The CYP3A5 polymorphism has been reported to affect the pharmacokinetics of alprazolam, but its effect on midazolam kinetics has been inconclusive. For etizolam and desmethylclobazam, some data suggest that CYP2C19 deficiency leads to side-effects or toxicity. For the remaining BZPs the clinical significance of the observed pharmacokinetic changes remains unclear. Further studies on the effects of genetic polymorphisms of CYP enzymes on the pharmacokinetics and pharmacodynamics of BZPs are necessary to guide treatment individualization and optimization. PMID:17635335

  9. Subcellular localization of pituitary enzymes

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  10. Micromotors Powered by Enzyme Catalysis.

    Dey, Krishna K; Zhao, Xi; Tansi, Benjamin M; Méndez-Ortiz, Wilfredo J; Córdova-Figueroa, Ubaldo M; Golestanian, Ramin; Sen, Ayusman

    2015-12-01

    Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture. PMID:26587897

  11. Human cytochrome P450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes

    Jeurissen, S.M.F.; Punt, A.; Boersma, M.G.; Bogaards, J.J.P.; Fiamegos, Y.C.; Schilter, B.; Bladeren, P.J. van; Cnubben, N.H.P.; Rietjens, I.M.C.M.

    2007-01-01

    Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1′-hydroxyestragole were identified and compared to the enzymes of importance for 1′-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that

  12. Proteomic and activity profiles of ascorbate-glutathione cycle enzymes in germinating barley embryo

    Bønsager, Birgit Christine; Shahpiri, Azar; Finnie, Christine;

    2010-01-01

    Enzymes involved in redox control are important during seed germination and seedling growth. Ascorbate-glutathione cycle enzymes in barley embryo extracts were monitored both by 2D-gel electrophoresis and activity measurements from 4 to 144 h post imbibition (PI). Strikingly different activity...

  13. Proteinaceous inhibitors of carbohydrate-active enzymes in cereals: implication in agriculture, cereal processing and nutrition

    Juge, N.; Svensson, Birte

    2006-01-01

    Enzymes that degrade, modify, or create glycosidic bonds are involved in carbohydrate biosynthesis and remodelling. Microbial carbohydrate-active enzymes form the basis of current green technology in the food, feed, starch, paper and pulp industries and the revolution in genomics may offer long...

  14. Nuclear quantum effects and kinetic isotope effects in enzyme reactions.

    Vardi-Kilshtain, Alexandra; Nitoker, Neta; Major, Dan Thomas

    2015-09-15

    Enzymes are extraordinarily effective catalysts evolved to perform well-defined and highly specific chemical transformations. Studying the nature of rate enhancements and the mechanistic strategies in enzymes is very important, both from a basic scientific point of view, as well as in order to improve rational design of biomimetics. Kinetic isotope effect (KIE) is a very important tool in the study of chemical reactions and has been used extensively in the field of enzymology. Theoretically, the prediction of KIEs in condensed phase environments such as enzymes is challenging due to the need to include nuclear quantum effects (NQEs). Herein we describe recent progress in our group in the development of multi-scale simulation methods for the calculation of NQEs and accurate computation of KIEs. We also describe their application to several enzyme systems. In particular we describe the use of combined quantum mechanics/molecular mechanics (QM/MM) methods in classical and quantum simulations. The development of various novel path-integral methods is reviewed. These methods are tailor suited to enzyme systems, where only a few degrees of freedom involved in the chemistry need to be quantized. The application of the hybrid QM/MM quantum-classical simulation approach to three case studies is presented. The first case involves the proton transfer in alanine racemase. The second case presented involves orotidine 5'-monophosphate decarboxylase where multidimensional free energy simulations together with kinetic isotope effects are combined in the study of the reaction mechanism. Finally, we discuss the proton transfer in nitroalkane oxidase, where the enzyme employs tunneling as a catalytic fine-tuning tool. PMID:25769515

  15. RENAL INVOLVEMENT IN LEPTOSPIROSIS

    Galya I. Gancheva

    2012-01-01

    Renal involvement is a common feature of leptospirosis. It is variable from mild to severe acute renal failure. Materials and methods: We performed analysis of 100 consecutive leptospirosis cases treated in Clinic of Infectious Diseases at University Hospital – Pleven (1976-2012) (90 male, age 37±18 years, lethal outcome in 13%), followed by comparative analysis of group with renal involvement (n1=59) versus group without renal involvement (controls: n2=41). Results: Fever (100%), hepatomegal...

  16. A DNA enzyme with N-glycosylase activity

    Sheppard, Terry L.; Ordoukhanian, Phillip; Joyce, Gerald F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 106-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of...

  17. The pharmacogenetics of cytochrome P450 enzymes in personalized medicine

    Majid Moridani

    2007-06-01

    Full Text Available Personalized medicine is partially enabled by in vitro diagnostics including pharmacogenomic, proteomic and other functional testing such as therapeutic drug management and toxicological testing. This paper will introduce the conceptual aspects of developing personalized treatment using pharmacogenetics information. The initial discussion will give an overview of the application of pharmacogenetics in personalized medicine, followed by specific examples involving cytochrome P450s drug metabolizing enzymes (CYP enzymes. The paper also discusses the influence of racial and ethnic characteristics of a population on the variation in drug effectiveness and toxicity. The need for implementation of pharmacogenetics in medical education is highlighted.

  18. The action of enzymes on rhodopsin.

    RADDING, C M; WALD, G

    1958-11-20

    The effects have been examined of chymotrypsin, pepsin, trypsin, and pancreatic lipase on cattle rhodopsin in digitonin solution. The digestion of rhodopsin by chymotrypsin was measured by the hydrolysis of peptide bonds (formol titration), changes in pH, and bleaching. The digestion proceeds in two stages: an initial rapid hydrolysis which exposes about 30 amino groups per molecule, without bleaching; superimposed on a slower hydrolysis which exposes about 50 additional amino groups, with proportionate bleaching. The chymotryptic action begins at pH about 6.0 and increases logarithmically in rate to pH 9.2. Trypsin and pepsin also bleach rhodopsin in solution. A preparation of pancreatic lipase bleached it slightly, but no more than could be explained by contamination with proteases. In digitonin solution each rhodopsin molecule is associated in a micelle with about 200 molecules of digitonin; yet the latter do not appear to hinder enzyme action. It is suggested that the digitonin sheath is sufficiently fluid to be penetrated on collision with an enzyme molecule; and that once together the enzyme and substrate are held together by intermolecular attractive forces, and by the "cage effect" of bombardment by surrounding solvent molecules. The two stages of chymotryptic digestion of rhodopsin may correspond to an initial rapid fragmentation, such as has been observed with many proteinases and substrates; superimposed upon a slower digestion of the fragments. Since the first phase involves no bleaching, this may mean that rhodopsin can be broken into considerably smaller fragments without loss of optical properties. PMID:13587919

  19. Inhibition of Human Cytochrome P450 Enzymes by Bacopa monnieri Standardized Extract and Constituents

    Seetha Ramasamy

    2014-02-01

    Full Text Available Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL, CYP2C9 (36.49/12.5 µg/mL, CYP1A2 (52.20/25.1 µg/mL; competitive inhibition of CYP3A4 (83.95/14.5 µg/mL and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL. However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day, B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%. These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19.

  20. In vitro metabolism of the mycotoxin enniatin B in different species and cytochrome p450 enzyme phenotyping by chemical inhibitors.

    Fæste, Christiane K; Ivanova, Lada; Uhlig, Silvio

    2011-09-01

    Enniatins are cyclic hexapeptidic mycotoxins produced by fungi growing on field grains, especially in wet climates. They show considerable resistance to food and feed processing technologies and might cause intoxication of humans and animals. Enniatins are also under exploration as anticancer drugs. The observed difference of in vitro and in vivo toxicities suggests low absorption or fast elimination of the enniatins after oral uptake. In the study presented here, in vitro metabolism studies of enniatin B were performed using rat, dog, and human liver microsomes under conditions of linear kinetics to estimate the respective elimination rates. Furthermore, cytochrome P450 reaction phenotyping with chemical inhibitors selective for human enzymes was carried out. Twelve metabolites were separated and characterized by multiple high-performance liquid chromatographic/mass spectrometric analyses as products of oxidation and demethylation reactions. Biotransformation rates and metabolite patterns varied considerably in the three species. The intrinsic clearances determined in assays with rat, dog, and human liver microsomes were 1.16, 8.23, and 1.13 l/(h · kg), respectively. The predicted enniatin B in vivo blood clearances were 1.57 l/(h · kg) in rats, 1.67 l/(h · kg) in dogs, and 0.63 l/(h · kg) in humans. CYP3A4 was important for enniatin B metabolism in human microsomes as shown by 80% inhibition and impaired metabolite formation in the presence of troleandomycin. CYP1A2 and CYP2C19 were additionally involved. Preliminary results showed that CYP3A and CYP1A might also be relevant in rats and dogs. The extensive hepatic metabolism could explain the reduced in vivo potential of enniatin B. PMID:21622627

  1. Involved Node Radiation Therapy

    Maraldo, Maja V; Aznar, Marianne C; Vogelius, Ivan R;

    2012-01-01

    PURPOSE: The involved node radiation therapy (INRT) strategy was introduced for patients with Hodgkin lymphoma (HL) to reduce the risk of late effects. With INRT, only the originally involved lymph nodes are irradiated. We present treatment outcome in a retrospective analysis using this strategy ...

  2. Eye Involvement in TSC

    ... of the eyes) should prompt inquiries into the family history and TSC involvement. Also, ophthalmologists should remember that mental handicap is ... or depigmented area and should inquire about the family’s history and any possible involvement in TSC. **This publication from the Tuberous Sclerosis ...

  3. Thermodynamics of Enzyme-Catalyzed Reactions Database

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  4. Modifying enzyme activity and selectivity by immobilization

    Rodrigues, Rafael C.; Ortiz, Claudia; Berenguer Murcia, Ángel; Torres, Rodrigo; Fernández Lafuente, Roberto

    2013-01-01

    Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will ...

  5. Udfordringer ved undervisning i enzymer

    Skriver, Karen; Dandanell, Gert; von Stemann, Jakob Hjorth;

    2015-01-01

    Enzymer er et centralt emne i biokemiundervisning. Det forudsætter og anvender grundlæggende viden inden for og kompetencer i kemi og matematik. Artiklen undersøger hvilke forståelsesvanskeligheder og udfordringer der er knyttet til dette område, såvel som virtuelle øvelsers potentiale i denne...

  6. Insolubilized enzymes for food synthesis

    Marshall, D. L.

    1972-01-01

    Cellulose matrix with numerous enzyme-coated silica particles of colloidal size permanently bound at various sites within matrix was produced that has high activity and possesses requisite physical characteristics for filtration or column operations. Product also allows coupling step in synthesis of edible food to proceed under mild conditions.

  7. The chondroitin/dermatan sulfate synthesizing and modifying enzymes in laryngeal cancer: Expressional and epigenetic studies

    Kalathas, Dimitrios; Triantaphyllidou, Irene-Eva; Mastronikolis, Nicholas S; Goumas, Panos D; Papadas, Thoedore A; Tsiropoulos, Gabriel; Vynios, Demitrios H.

    2010-01-01

    Background Significant biochemical changes are observed in glycosaminoglycans in squamous cell laryngeal carcinoma. The most characteristics are in chondroitin/dermatan sulfate fine structure and proportion, which might be due to differential expression of the enzymes involved in their biosynthesis. The aim of the present work was the investigation in expressional and epigenetic level of the enzymes involved in chondroitin/dermatan sulfate biosynthesis in laryngeal cancer. Methods Tissues sub...

  8. 褪黑素对大鼠肝微粒体细胞色素P450酶亚型CYP2C9、CYP2D6、CYP3A4活性的影响

    刘明远; 杨光远; 王跃新; 张春斌; 白雪; 张建华

    2011-01-01

    目的 观察褪黑素对大鼠肝微粒体内细胞色素P450 (CYP450) 酶亚型CYP2C9、CYP2D6、CYP3A4活性的影响.方法 以生理盐水为对照,大鼠灌胃0.54 mg·kg-1·d-1的褪黑素,连续7 d,然后测定其肝微体中CYP2C9、CYP2D6、CYP3A4活性.结果 与对照组比较,褪黑素组CYP2C9、CYP2D6、CYP3A4的活性无变化(P>0.05).结论 褪黑素对CYP450酶 CYP2C9、CYP2D6、CYP3A4活性无影响.

  9. PROTEIN-PROTEIN INTERACTIONS OF THE FLAVONOID BIOSYNTHETIC ENZYMES IN ARABIDOPSIS THALIANA

    Xiang, Jiale

    2014-01-01

    Flavonoids are a group of secondary metabolites, which are not only important for plants’ survival, but also have been found to have medicinal properties for human health. Several enzymes are involved in the flavonoid biosynthesis. It is thought that these enzymes work together and may form enzymatic complexes. But the way of these enzymes interact with each other is still not clear. In arabidopsis, the number of gene family members that encode these enzymes is less than in other model plan...

  10. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  11. Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids.

    Romanov, V I; Hernández-Lucas, I; Martínez-Romero, E

    1994-07-01

    We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids. PMID:16349319

  12. Enzymes and bioproducts produced by the ascomycete fungus Paecilomyces variotii.

    Herrera Bravo de Laguna, I; Toledo Marante, F J; Mioso, R

    2015-12-01

    Due its innate ability to produce extracellular enzymes which can provide eco-friendly solutions for a variety of biotechnological applications, Paecilomyces variotii is a potential source of industrial bioproducts. In this review, we report biotechnological records on the biochemistry of different enzymes produced by the fermentation of the P. variotii fungus, including tannases, phytases, cellulases, xylanases, chitinases, amylases and pectinases. Additionally, the main physicochemical properties which can affect the enzymatic reactions of the enzymes involved in the conversion of a huge number of substrates to high-value bioproducts are described. Despite all the background information compiled in this review, more research is required to consolidate the catalytic efficiency of P. variotii, which must be optimized so that it is more accurate and reproducible on a large scale. PMID:26274842

  13. An NMR investigation of isotope exchange involving multiply labelled intermediates

    Information about enzyme systems can be obtained from studies of isotope exchange reactions. NMR can monitor these reactions continuously, even in intact biological samples. In this paper 1H NMR is used to study a 1H/2H exchange process which involves multiple labelling of NADH. The NADH is an intermediate in an exchange process involving the glycolytic enzymes glyceraldehyde phosphate dehydrogenase, lactate dehydrogenase, triose phosphate isomerase and aldolase. The form of the observed exchange is predicted by an extended theory of isotope exchange. (author)

  14. Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soil enzymes originate from a variety of organisms, notably fungi and bacteria...... and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity reflects the functional diversity and activity of the microorganisms involved in decomposition processes which are essential processes for soil...... functioning and soil ecosystem services. The soil enzyme activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysaccharides as cellulose, hemicellulose and chitin, while degradation of proteins...

  15. Temporal changes in glycogenolytic enzyme mRNAs during myogenesis of primary porcine satellite cells

    Henckel, Poul; Theil, Peter Kappel; Sørensen, Inge Lise; Oksbjerg, Niels

    2007-01-01

    , phosphorylase kinase, phosphorylase and glycogen debranching enzyme, and no alterations of the transporter molecule GLUT4, clearly indicate that glycogenolytic enzymes of potential importance to meat quality development are regulated at the gene level during myogenesis, and are heavily involved in muscle cell...... and muscle fibre development. The genes, however, are not influenced by insulin, and the lack of response to insulin of expression of gene-encoding enzymes involved in the formation and degradation of glycogen may question the applicability of porcine cell culture systems, like the one applied, as a...

  16. Doctors' involvement in torture

    Sonntag, Jesper

    2008-01-01

    Doctors from both non-democratic and democratic countries are involved in torture. The majority of doctors involved in torture are doctors at risk. Doctors at risk might compromise their ethical duty towards patients for the following possible reasons: individual factors (such as career, economic...... or ideological reasons), threats, orders from a higher ranking officer, political initiatives, working in atrocity-producing situations or dual loyalty. In dual loyalty conflicts, factors that might compromise doctors' ethical obligations towards detainees/patients are: ideological totalitarianism......, moral disengagement, victim blame, patriotism, individual factors or threats. Another important reason why doctors are involved in torture is that not all doctors are trained in addressing human rights issues of detainees. Torture survivors report that they have experienced doctors' involvement in...

  17. Bedaquiline Metabolism: Enzymes and Novel Metabolites

    Liu, Ke; Li, Feng; Lu, Jie; Liu, Shinlan; Dorko, Kenneth; Xie, Wen; Ma, Xiaochao

    2014-01-01

    Bedaquiline is a recently approved drug for the treatment of multidrug-resistant tuberculosis. Adverse cardiac and hepatic drug reactions to bedaquiline have been noted in clinical practice. The current study investigated bedaquiline metabolism in human hepatocytes using a metabolomic approach. Bedaquiline N-demethylation via CYP3A4 was confirmed as the major pathway in bedaquiline metabolism. In addition to CYP3A4, we found that both CYP2C8 and CYP2C19 contributed to bedaquiline N-demethylat...

  18. Botryosphaeriales fungi produce extracellular enzymes with biotechnological potential.

    Esteves, Ana Cristina; Saraiva, Márcia; Correia, António; Alves, Artur

    2014-05-01

    Phytopathogenic fungi are known for producing an arsenal of extracellular enzymes whose involvement in the infection mechanism has been suggested. However, these enzymes are largely unknown and their biotechnological potential also remains poorly understood. In this study, the production and thermostability of extracellular enzymes produced by phytopathogenic Botryosphaeriaceae was investigated. Hydrolytic and oxidative activities were detected and quantified at different temperatures. Most strains (70%; 37/53) were able to produce simultaneously cellulases, laccases, xylanases, pectinases, pectin lyases, amylases, lipases, and proteases. Surprisingly for mesophilic filamentous fungi, several enzymes proved to be thermostable: cellulases from Neofusicoccum mediterraneum CAA 001 and from Dothiorella prunicola CBS 124723, lipases from Diplodia pinea (CAA 015 and CBS 109726), and proteases from Melanops tulasnei CBS 116806 were more active at 70 °C than at any of the other temperatures tested. In addition, lipases produced by Diplodia pinea were found to be significantly more active than any other known lipase from Botryosphaeriales. The thermal activity profile and the wide array of activities secreted by these fungi make them optimal producers of biotechnologically relevant enzymes that may be applied in the food and the health industries (proteases), the pulp-and-paper and biofuel industries (cellulases), or even in the detergent industry (lipases, proteases, amylases, and cellulases). PMID:24802941

  19. Perspectives in developing industrial enzymes by using technological intelligence

    Óscar Fernando Castellanos Domínguez

    2010-04-01

    Full Text Available Developing emergent technologies implies an ongoing challenge involving using new technological management tools. Enzyme engineering and its respective technology (within the context of biotechnology is one of the areas of knowledge from which great expectations are constantly arising, such as providing competitive advantage in emergent economies like that of Colombia. However, several decades of research in this field have still not led to important results in terms of enzyme production and their application in industrial processes. This article proposes applying the components of a technological intelligence system to developing enzyme technology. Initial emphasis is placed on scientometrics, using exercises for mapping patents, technological monitoring and evaluating Colombian prolects forming part of Colciencias’ ScienTI network, carried out for the enzymes being used in both the food industry for obtaining hydrolysed products and in the textile industry. An example of Delphi study is documented within the context of industrial enzymes’ future within biotechnology in general. The learning which can arise from analysing a sector leader is stressed by using organisational benchmarking. The basis of a technological intelligence system is then discussed in terms of how it can provide productive and competitive strategies aimed at applying industrial enzymes.

  20. SKPDB: a structural database of shikimate pathway enzymes

    de Azevedo Walter F

    2010-01-01

    Full Text Available Abstract Background The functional and structural characterisation of enzymes that belong to microbial metabolic pathways is very important for structure-based drug design. The main interest in studying shikimate pathway enzymes involves the fact that they are essential for bacteria but do not occur in humans, making them selective targets for design of drugs that do not directly impact humans. Description The ShiKimate Pathway DataBase (SKPDB is a relational database applied to the study of shikimate pathway enzymes in microorganisms and plants. The current database is updated regularly with the addition of new data; there are currently 8902 enzymes of the shikimate pathway from different sources. The database contains extensive information on each enzyme, including detailed descriptions about sequence, references, and structural and functional studies. All files (primary sequence, atomic coordinates and quality scores are available for downloading. The modeled structures can be viewed using the Jmol program. Conclusions The SKPDB provides a large number of structural models to be used in docking simulations, virtual screening initiatives and drug design. It is freely accessible at http://lsbzix.rc.unesp.br/skpdb/.

  1. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  2. Lithuanian biochemist builds enzyme empire

    Dickman, S.

    1992-09-11

    Vidas Janulaitis is professor of biochemistry at the University of Vilnius, head of the Institute of Applied Enzymology - and creator of one of the world's largest collections of restriction enzymes, with more than 100 on offer. He also appears to be the first successful biotechnology entrepreneur to emerge from the former Soviet Union. This paper shows how Janulaitis managed to rise above the chaos that has accompanied the dismantlement of the Soviet Union to become one of the world's top suppliers of new restriction enzymes - especially given that the venture capitalists who rushed off to make deals with Moscow labs in the early days of perestroika mostly came back disappointed.

  3. Metrological aspects of enzyme production

    Kerber, T. M.; Dellamora-Ortiz, G. M.; Pereira-Meirelles, F. V.

    2010-05-01

    Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies.

  4. Metrological aspects of enzyme production

    Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies

  5. Enzyme recovery using reversed micelles.

    Dekker, M.

    1990-01-01

    The objective of this study was to develop a liquid-liquid extraction process for the recovery of extracellular enzymes. The potentials of reaching this goal by using reversed micelles in an organic solvent have been investigated.Reversed micelles are aggregates of surfactant molecules containing an inner core of water molecules, dispersed in a continuous organic solvent medium. The considerable biotechnological potential of these systems is derived principally from the ability of the water d...

  6. Immobilised enzymes in biorenewable production

    Franssen, M.C.R.; Steunenberg, P.; Scott, E.L.; Zuilhof, H.; Sanders, J.P.M.

    2013-01-01

    Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, ...

  7. Substrate mediated enzyme prodrug therapy.

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  8. Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng.

    Jian Xiao

    Full Text Available Ginsenoside compound K (CK, a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The Km and Vmax values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC50 values were 16.00 μM and 9.83 μM, and Ki values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.

  9. Pancreatic Involvement in Melioidosis

    Vui Heng Chong

    2010-07-01

    Full Text Available Context Melioidosis is endemic to tropical regions and, despite the common occurrence of intra-abdominal abscesses, pancreatic involvement in melioidosis has not previously been reported. Objective We report our experience with pancreatic melioidosis. Patients All 65 patients treated for melioidosis who had computed tomography (CT scans were identified from prospective databases and were retrospectively reviewed. Main outcome measures A detailed review of cases with pancreas involvement was carried out. Results There were four cases (three males and one female; median age 29.5 years, range: 25-48 years with pancreatic melioidosis, giving a prevalence of 6.2%. All had predisposing conditions (two had poorly controlled diabetes mellitus and two had thalassemia for melioidosis. Fever (100%, anorexia (100%, weight loss (100%, rigor (75% and abdominal pain (75% were the most common symptoms at presentation and the median duration of symptoms before presentation was six weeks (range: 2-8 weeks. All pancreatic abscesses were detected on CT scan. Multiple foci involvement was common (3 to 6 sites: blood (4 patients, liver (3 patients, psoas muscle (2 patients, spleen (2 patients, infected ascites (2 patients and lung (1 patient. Pancreatic involvement ranged from multi-focal micro-abscesses to focal large abscesses and involved all parts of the pancreas (body 100%, head 75% and tail 50%. Associated pancreatic findings included splenic vein thrombosis, peripancreatic inflammation and peripancreatic fat streaking. All the pancreatic abscesses were resolved with antibiotics without requiring pancreatic abscess drainage (including one patient who died from disseminated melioidosis. Conclusion Pancreatic involvement typically occurs as part of multi-organ involvement and commonly manifests as multifoci micro-abscesses. Associated pancreatic abnormalities were also common. All responded to treatment without requiring drainage

  10. Determination of plasma gluthatione reductase enzyme activity in osteoporotic women

    Sadeghi N; Oveisi M.R.; Jannat B.; Hajimahmoodi M; Jamshidi A.R; Sajadian Z.

    2008-01-01

    Background: Osteoporosis is a disease of high prevalence with increased bone loss. Free radicals have been proved to be involved in bone resorption. Glutathione reductase (GR) plays an essential role in cell defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant, glutathione. In the present study GR activity of plasma as an antioxidant enzyme in relation to Bone Mineral Density (BMD) was investigated.Material and Method: GR activity was measur...

  11. Extracellular enzyme kinetics scale with resource availability

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...

  12. Involvement, Tate and me

    Slater, Alix; Armstrong, Kate

    2010-01-01

    The involvement construct has been explored in relation to products, services and leisure but not in an art museum context. The purpose of this paper is to address this theoretical gap by drawing on the marketing and leisure literature to understand members’ consumption of Tate using the involvement construct. Tate, a portfolio of four art museums in the UK has more than 90,000 members that receive a benefits package in return for a membership fee. Data were collected using an interpretive, q...

  13. The Application of Enzyme and Yeast

    Zhao, Qing

    2012-01-01

    This bachelor’s thesis concerns the application of enzymes and yeasts for bio-industry. The purpose of this work is to understand the basic knowledge about enzyme and yeast, and meanwhile, to find out their different applications. Through comprehensive study, the knowledge was accumulated which brought a clear understanding for the enzyme structure and yeast microorganism, together with their working principles for the bioprocess. For wood-based industry, the different enzymes used in bi...

  14. 21 CFR 864.4400 - Enzyme preparations.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme preparations. 864.4400 Section 864.4400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the...

  15. The ENZYME data bank in 1999.

    Bairoch, A

    1999-01-01

    The ENZYME data bank is a repository of information related to the nomenclature of enzymes. In recent years it has become an indispensable resource for the development of metabolic databases. The current version contains information on 3704 enzymes. It is available through the ExPASy WWW server (http://www.expasy.ch/). PMID:9847212

  16. The ENZYME data bank in 1995.

    Bairoch, A

    1996-01-01

    The ENZYME data bank is a repository of information relative to the nomenclature of enzymes. The current version (October 1995) contains information relevant to 3594 enzymes. It is available from a variety of file and ftp servers as well as through the ExPASy World Wide Web server (http://expasy.hcuge.ch/). PMID:8594586

  17. Getting Parents Involved.

    Butts, Vickie; Finch, Patty A.

    1985-01-01

    Describes a parental involvement program in reading, writing, and human education. The project consists of caring for Clifford, a stuffed toy dog, on a rotated basis by first grade students. Books and pet care items accompany Clifford and provide an opportunity for parent and child to work together. (ML)

  18. Curious cases of the enzymes

    Ulusu, Nuriye Nuray

    2015-01-01

    Life as we know it heavily relies on biological catalysis, in fact, in a very nonromantic version of it, life could be considered as a series of chemical reactions, regulated by the guarding principles of thermodynamics. In ancient times, a beating heart was a good sign of vitality, however, to me, it is actually the presence of active enzymes that counts. Though we do not usually pay attention, the history of enzymology is as old as humanity itself, and dates back to the ancient times. This ...

  19. Curious cases of the enzymes

    Ulusu Nuriye Nuray

    2015-01-01

    J Med Biochem 2015; 34 (3) DOI: 10.2478/jomb-2014-0045 UDK 577. 1 : 61 ISSN 1452-8258 J Med Biochem 34: 271–281, 2015 Review article Pregledni ~lanak CURIOUS CASES OF THE ENZYMES NEOBI^NA ISTORIJA ENZIMA Nuriye Nuray Ulusu Koç University, School of Medicine, Sariyer-Istanbul, Turkey Address for correspondence: N. Nuray Ulusu, PhD Koç University School of Medicine Professor of Biochemistry Rumelifeneri Yolu Sarıyer-Istanbul – Turkey Phone: +90 (212)...

  20. Consumer attitudes to enzymes in food production

    Søndergaard, Helle Alsted; Grunert, Klaus G.; Scholderer, Joachim

    2005-01-01

    The use of enzymes in food production has potential benefits for both food manufacturers and consumers. A central question is how consumers react to new ways of producing foods with enzymes. This study investigates the formation of consumer attitudes to different enzyme production methods in three...... European countries. Results show that consumers are most positive towards non-GM enzyme production methods. The enzyme production method is by far the most important factor for the formation of buying intentions compared to price and benefits. Results also show that environmental concern and attitudes...

  1. Involving the Young

    Walter Hirche

    2012-01-01

    Youth unemployment is a huge challenge for sustainable development in our societies. Statistics offer an alarming picture of the involvement of youth in the labour markets on a global scale. Germany, due to a strong tradition in vocational education and training deeply rooted in its culture, has very low rates of youth unemployment. The so-called dual system of vocational training in Germany takes up essential features of the holistic approach in education which is fundamental for education f...

  2. DNA-Based Enzyme Reactors and Systems

    Veikko Linko

    2016-07-01

    Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

  3. Enzyme extraction by ultrasound from sludge flocs

    YU Guanghui; HE Pinjing; SHAO Liming; ZHU Yishu

    2009-01-01

    Enzymes play essential roles in the biological processes of sludge treatment. In this article, the ultrasound method to extract enzymes from sludge flocs was presented. Results showed that using ultrasound method at 20 kHz could extract more types of enzymes than that ultrasound at 40 kHz and ethylenediamine tetraacetic acid (EDTA) methods. The optimum parameters of ultrasound extraction at 20 kHz were duration of 10 min and power of 480 W. Under the condition, ultrasound could break the cells and extract both the extracellular and intercellular enzymes. Ultrasound power was apparently more susceptive to enzyme extraction than duration, suggesting that the control of power during ultrasound extraction was more important than that of duration. The Pearson correlation analysis between enzyme activities and cation contents revealed that the different types of enzymes had distinct cation binding characteristics.

  4. Involvement of hyaluronidases in colorectal cancer

    Hyaluronidases belong to a class of enzymes that degrade, predominantly, hyaluronan. These enzymes are known to be involved in physiological and pathological processes, such as tumor growth, infiltration and angiogenesis, but their exact role in tumor promotion or suppression is not clear yet. Advanced colorectal cancer is associated with elevated amounts of hyaluronan of varying size. The aim of the present study was therefore to illuminate the importance of hyaluronidases in colon carcinoma progression. The patients' samples (macroscopically normal and cancerous) were subjected to sequential extraction with PBS, 4 M GdnHCl and 4 M GdnHCl - 1% Triton X-100. The presence of the various hyaluronidases in the extracts was examined by zymography and western blotting. Their expression was also examined by RT-PCR. Among hyaluronidases examined, Hyal-1, -2, -3 and PH-20 were detected. Their activity was higher in cancerous samples. Hyal-1 and Hyal-2 were overexpressed in cancerous samples, especially in advanced stages of cancer. Both isoforms were mainly extracted with PBS. Hyal-3 was observed only in the third extract of advanced stages of cancer. PH-20 was abundant in all three extracts of all stages of cancer. The expression of only Hyal-1 and PH-20 was verified by RT-PCR. A high association of hyaluronidases in colorectal cancer was observed. Each hyaluronidase presented different tissue distribution, which indicated the implication of certain isoforms in certain cancer stages. The results provided new evidence on the mechanisms involved in the progression of colorectal cancer

  5. Identification of biotransformation enzymes in the antennae of codling moth Cydia pomonella.

    Huang, Xinglong; Liu, Lu; Su, Xiaoji; Feng, Jinian

    2016-04-10

    Biotransformation enzymes are found in insect antennae and play a critical role in degrading xenobiotics and odorants. In Cydia pomonella, we identified 26 biotransformation enzymes. Among these enzymes, twelve carboxylesterases (CXEs), two aldehyde oxidases (AOXs) and six alcohol dehydrogenases (ADs) were predominantly expressed in antennae. Each of the CpomCXEs presents a conserved catalytic triad "Ser-His-Glu", which is the structural characteristic of known insect CXEs. CpomAOXs present two redox centers, a FAD-binding domain and a molybdenum cofactor/substrate-binding domain. The antennal CpomADs are from two protein families, short-chain dehydrogenases/reducetases (SDRs) and medium-chain dehydrogenases/reducetases (MDRs). Putative catalytic active domain and cofactor binding domain were found in these CpomADs. Potential functions of these enzymes were determined by phylogenetic analysis. The results showed that these enzymes share close relationship with odorant degrading enzymes (ODEs) and resistance-associated enzymes of other insect species. Because of commonly observed roles of insect antennal biotransformation enzymes, we suggest antennal biotransformation enzymes presented here are candidate that involved in degradation of odorants and xenobiotics within antennae of C. pomonella. PMID:26778204

  6. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    Urban, Philippe; Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate ...

  7. Inhibitors of Testosterone Biosynthetic and Metabolic Activation Enzymes

    Leping Ye

    2011-12-01

    Full Text Available The Leydig cells of the testis have the capacity to biosynthesize testosterone from cholesterol. Testosterone and its metabolically activated product dihydrotestosterone are critical for the development of male reproductive system and spermatogenesis. At least four steroidogenic enzymes are involved in testosterone biosynthesis: Cholesterol side chain cleavage enzyme (CYP11A1 for the conversion of cholesterol into pregnenolone within the mitochondria, 3β-hydroxysteroid dehydrogenase (HSD3B, for the conversion of pregnenolone into progesterone, 17α-hydroxylase/17,20-lyase (CYP17A1 for the conversion of progesterone into androstenedione and 17β-hydroxysteroid dehydrogenase (HSD17B3 for the formation of testosterone from androstenedione. Testosterone is also metabolically activated into more potent androgen dihydrotestosterone by two isoforms 5α-reductase 1 (SRD5A1 and 2 (SRD5A2 in Leydig cells and peripheral tissues. Many endocrine disruptors act as antiandrogens via directly inhibiting one or more enzymes for testosterone biosynthesis and metabolic activation. These chemicals include industrial materials (perfluoroalkyl compounds, phthalates, bisphenol A and benzophenone and pesticides/biocides (methoxychlor, organotins, 1,2-dibromo-3-chloropropane and prochloraz and plant constituents (genistein and gossypol. This paper reviews these endocrine disruptors targeting steroidogenic enzymes.

  8. Psychrophilic Enzymes: From Folding to Function and Biotechnology

    Georges Feller

    2013-01-01

    Full Text Available Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted.

  9. Optimization to Low Temperature Activity in Psychrophilic Enzymes

    Caroline Struvay

    2012-09-01

    Full Text Available Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts.

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

    Miao Hao

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

  11. Promoting vital involvement.

    Kivnick, Helen Q; Stoffel, Sharon A

    2002-09-01

    Health care for the elderly generally focuses on health problems. This approach ignores the strengths and resources that maximize a person's autonomy, integrity, and ability to make contributions to society; and it exacerbates poor health. Vital involvement practice (VIP) is an approach to caring for the elderly that emphasizes an individual's capabilities by exploring factors both internal and external to the individual. VIP is identified as a model for health care providers that will improve the health and quality of life of elderly patients. PMID:12387120

  12. The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes

    We have measured cytochrome P450 (CYP) activity in nearly 150 samples of human liver microsomes and 64 samples of cryopreserved human hepatocytes, and we have performed induction studies in over 90 preparations of cultured human hepatocytes. We have analyzed these data to examine whether the expression of CYP enzyme activity in liver microsomes and isolated hepatocytes or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age, or ethnicity of the donor (the latter being limited to Caucasians, African Americans, and Hispanics due to a paucity of livers from Asian donors). In human liver microsomes, there were no statistically significant differences (P > 0.05) in CYP activity as a function of age, gender, or ethnicity with one exception. 7-Ethoxyresorufin O-dealkylase (CYP1A2) activity was greater in males than females, which is consistent with clinical observation. Liver microsomal testosterone 6β-hydroxylase (CYP3A4) activity was slightly greater in females than males, but the difference was not significant. However, in cryopreserved human hepatocytes, the gender difference in CYP3A4 activity (females = twice males) did reach statistical significance, which supports the clinical observation that females metabolize certain CYP3A4 substrates faster than do males. Compared with those from Caucasians and African Americans, liver microsomes from Hispanics had about twice the average activity of CYP2A6, CYP2B6, and CYP2C8 and half the activity of CYP1A2, although this apparent ethnic difference may be a consequence of the relatively low number of Hispanic donors. Primary cultures of hepatocytes were treated with β-naphthoflavone, an inducer of CYP1A2, phenobarbital or rifampin, both of which induce CYP2B6, CYP2C9, CYP2C19, and CYP3A4, albeit it to different extents. Induction of these CYP enzymes in freshly cultured hepatocytes did not appear to be influenced by the gender or age of the donor. Furthermore, CYP3A4 induction in

  13. Use of Human Plasma Samples to Identify Circulating Drug Metabolites that Inhibit Cytochrome P450 Enzymes.

    Eng, Heather; Obach, R Scott

    2016-08-01

    Drug interactions elicited through inhibition of cytochrome P450 (P450) enzymes are important in pharmacotherapy. Recently, greater attention has been focused on not only parent drugs inhibiting P450 enzymes but also on possible inhibition of these enzymes by circulating metabolites. In this report, an ex vivo method whereby the potential for circulating metabolites to be inhibitors of P450 enzymes is described. To test this method, seven drugs and their known plasma metabolites were added to control human plasma at concentrations previously reported to occur in humans after administration of the parent drug. A volume of plasma for each drug based on the known inhibitory potency and time-averaged concentration of the parent drug was extracted and fractionated by high-pressure liquid chromatography-mass spectrometry, and the fractions were tested for inhibition of six human P450 enzyme activities (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Observation of inhibition in fractions that correspond to the retention times of metabolites indicates that the metabolite has the potential to contribute to P450 inhibition in vivo. Using this approach, norfluoxetine, hydroxyitraconazole, desmethyldiltiazem, desacetyldiltiazem, desethylamiodarone, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion were identified as circulating metabolites that inhibit P450 activities at a similar or greater extent as the parent drug. A decision tree is presented outlining how this method can be used to determine when a deeper investigation of the P450 inhibition properties of a drug metabolite is warranted. PMID:27271369

  14. Extraction and Purification of Collagenase Enzymes: A Critical Review

    Said M. Daboor

    2010-01-01

    Full Text Available Problem statement: Enzymes have vital roles in several industrial processes (foods, cosmetics, nutraceuticals and pharmaceuticals due to their highly selective nature and high activity at very low concentrations. Recent efforts to identify new sources of useful enzymes have been concentrated on the marine environment because of the potential to make use of processing wastes. About 35-50% of the mass of the fish caught is a waste that is disposed off at sea or in landfills. The extraction of enzymes from fish processing waste can reduce environment problems and improve the economics of the fish industry. Collagenases are a group of enzymes that can be extracted from fish waste. Approach: Comprehensive reviews of the literature on the extraction, purification, characterization and use of collagenases was carried out. Results: Collagenases have different molecular weights based on their types and sources. They have the ability to break down the peptide bonds in collagen at physiological pH. They are classified into two types: serine and metallocollagenase. Collagenolytic activities have been shown at a wide range of temperatures (20-40°C and pH (6-8. Many activators can be used to achive collagenase activity including 4-Aminophenylmercuric Acetate (APMA, trypsin, potassium or sodium thiocyanate, iodoacetamide and potassium iodide. Dithiothreitol (DTT, mercaptoethanol, ethylendiaminetetracetic acid, o-phenanthroline and cysteine inactivate the enzyme. Collagenases enzymes can be extracted with a variety of techniques using different buffering systems (tris-HCl, sodium bicarbonate, calcium chloride and cacodylate. All techniques involve the use of ammonium sulphate fractionation and centrifugation to precipitate the enzyme. Collagenases are normally purified using chromatographic techniques such as gel-filtration, ion-exchange and affinity column chromatography. Collagenase can be assayed with a number of methods, including: colorimetric absorbance

  15. Analysis of enzyme production by submerged culture of Aspergillus oryzae using whole barley.

    Masuda, Susumu; Kikuchi, Kaori; Matsumoto, Yuko; Sugimoto, Toshikazu; Shoji, Hiroshi; Tanabe, Masayuki

    2009-10-01

    We have reported on high enzyme production by submerged culture of Aspergillus kawachii using barley with the husk (whole barley). To elucidate the mechanism underlying this high enzyme production, we performed a detailed analysis. Aspergillus oryzae RIB40 was submerged-cultured using whole barley and milled whole barley. Enzyme production was analyzed in terms of changes in medium components and gene expression levels. When whole barley was used, high production of glucoamylase and alpha-amylase and high gene expression levels of these enzymes were observed. Low ammonium concentrations were maintained with nitrate ion uptake continuing into the late stage using whole barley. These findings suggest that the sustainability of nitrogen metabolism is related to high enzyme production, and that a mechanism other than that associated with the conventional amylase expression system is involved in this relationship. PMID:19809198

  16. Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.

    Chung, Ben C; Zhao, Jinshi; Gillespie, Robert A; Kwon, Do-Yeon; Guan, Ziqiang; Hong, Jiyong; Zhou, Pei; Lee, Seok-Yong

    2013-08-30

    MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg(2+) within the active site, and provide a structural basis of catalysis for this class of enzyme. PMID:23990562

  17. A study of overproduction and enhanced secretion of enzymes. Quarterly report

    Dashek, W.V.

    1993-09-01

    Wood decay within forests, a significant renewable photosynthetic energy resource, is caused primarily by Basidiomycetous fungi, e.g., white rot fungi. These organisms possess the ability to degrade lignin, cellulose and hemicellulose, the main organic polymers of wood. In the case of the white rot fungi, e.g., Coriolus versicolor, the capacity results from the fungus` ability to elaborate extracellular cellulolytic and ligninolytic enzymes. With regard to the latter, at least one of the enzymes, polyphenol oxidase (PPO) appears within a defined growth medium. This proposal focuses on the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. There are two major sections to the proposal: (1) overproduction of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electro microscopical techniques and (2) the biochemical/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO enzymes.

  18. Identification and Modulation of Esterases Involved in the Metabolism of Heroin

    Hauan, Karine Margrethe

    2014-01-01

    Heroin is the main abused opioid, and is causing most drug use related deaths in the European Union and in the USA. New treatment strategies for heroin addiction are needed, and an alternative could be modulation of the enzymes involved in the heroin metabolism with the objective of reducing the rewarding effects of the drug. Different esterases have been shown to be involved in the metabolism of heroin. However, little is known about the importance of these enzymes in the heroin metabolism i...

  19. Multi-enzyme Process Modeling

    Andrade Santacoloma, Paloma de Gracia

    The subject of this thesis is to develop a methodological framework that can systematically guide mathematical model building for better understanding of multi-enzyme processes. In this way, opportunities for process improvements can be identified by analyzing simulations of either existing...... in the scientific literature. Reliable mathematical models of such multi-catalytic schemes can exploit the potential benefit of these processes. In this way, the best outcome of the process can be obtained understanding the types of modification that are required for process optimization. An effective evaluation...... of these processes is achieved by applying a methodological framework which provides a systematic way of modeling, a structure, guidance, documentation and support to the modeler. The methodological framework developed here brings many benefits to multienzyme process modeling. This framework identifies generic...

  20. Ethanologenic Enzymes of Zymomonas mobilis

    Ingram, Lonnie O' Neal

    1999-03-01

    Zymomonas mobilis is a unique microorganism in being both obligately fermentative and utilizing a Entner-Doudoroff pathway for glycolysis. Glycolytic flux in this organism is readily measured as evolved carbon dioxide, ethanol, or glucose consumed and exceeds 1 {micro}mole glucose/min per mg cell protein. To support this rapid glycolysis, approximately 50% of cytoplasmic protein is devoted to the 13 glycolytic and fermentative enzymes which constitute this central catabolic pathway. Only 1 ATP (net) is produced from each glucose metabolized. During the past grant period, we have completed the characterization of 11 of the 13 glycolytic genes from Z. mobilis together with complementary but separate DOE-fimded research by a former post-dot and collaborator, Dr. Tyrrell Conway. Research funded in my lab by DOE, Division of Energy Biosciences can be divided into three sections: A. Fundamental studies; B. Applied studies and utility; and C. Miscellaneous investigations.