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Sample records for epoxide hydrolase inhibition

  1. Selective inhibition and selective induction of multiple microsomal epoxide hydrolases.

    PubMed

    Guenthner, T M

    1986-03-01

    The inhibition in vitro and induction in vivo of microsomal trans-stilbene oxide hydrolase have been studied. This microsomal epoxide hydrolase activity is distinguishable from the previously well-defined microsomal arene oxide hydrolase by a number of catalytic criteria. Two substituted chalcone oxides, 4-phenylchalcone oxide and 4'-phenylchalcone oxide, are potent inhibitors of microsomal trans-stilbene oxide hydrolase, but have no apparent activity against benzo[a]pyrene 4,5-oxide hydrolase. Conversely, compounds that are potent inhibitors of benzo[a]pyrene 4,5-oxide hydrolase, including styrene oxide, cyclohexene oxide, and trichloropropene oxide, inhibit microsomal trans-stilbene oxide hydrolase only at very high (millimolar) concentrations. The chalcone oxides inhibit microsomal trans-stilbene oxide hydrolase noncompetitively, and have micromolar or nanomolar affinity constants for the enzyme. Attempts were made to induce microsomal trans-stilbene oxide hydrolase in vivo. Compounds that induced microsomal benzo[a]pyrene 4,5-oxide hydrolase levels in mice did not simultaneously induce trans-stilbene oxide hydrolase levels. Clofibrate was an exception; it induced levels of both enzymes to a small but statistically significant degree. The two microsomal hydrolase activities have, therefore, very different catalytic sites and appear to be under separate genetic control. 4-Phenylchalcone oxide and 4'-phenylchalcone oxide are selective inhibitors of microsomal trans-stilbene oxide hydrolase and may prove to be very useful in assessing the involvement of this enzyme in the metabolism of endogenous or xenobiotic epoxides.

  2. Inhibiting an epoxide hydrolase virulence strategy protects CFTR**

    PubMed Central

    Bahl, Christopher D.; Hvorecny, Kelli L.; Bomberger, Jennifer M.; Stanton, Bruce A.; Hammock, Bruce D.; Morisseau, Christophe; Madden, Dean R.

    2015-01-01

    Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, Cif's mechanism of action has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. Here, we show that Cif's hydrolase activity is strictly required for its effects on CFTR. We also uncover a small-molecule inhibitor that protects this key component of the mucociliary defense system. Our results provide a basis for targeting Cif's distinctive virulence chemistry and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking. PMID:26136396

  3. Prediction of drug-drug interactions with carbamazepine-10,11-epoxide using a new in vitro assay for epoxide hydrolase inhibition.

    PubMed

    Rosa, Maria; Bonnaillie, Pierre; Chanteux, Hugues

    2016-12-01

    1. Carbamazepine is an antiepileptic drug which is metabolized by CYP3A4 into carbamazepine-10,11-epoxide. This metabolite is then detoxified by epoxide hydrolase. As carbamazepine-10,11-epoxide has been associated with neurotoxicity, it is critical to identify whether a new antiepileptic drug has the potential to inhibit epoxide hydrolase and therefore increase carbamazepine-10,11-epoxide plasma levels. 2. In this study, an in vitro assay was developed to evaluate epoxide hydrolase activity by using carbamazepine-10,11-epoxide as probe substrate. The ability of this assay to predict drug-drug interactions (DDI) at the epoxide hydrolase level was also investigated. 3. To this aim, known inhibitors of epoxide hydrolase for which in vivo data are available were used. Firstly, carbamazepine-10,11-epoxide hydrolase activity was determined in liver microsomes, cytosol and hepatocytes. Thereafter, the IC50 of epoxide hydrolase inhibitors (progabide, valproic acid, valpromide and valnoctamide) was determined in liver microsomes and hepatocytes. Finally, prediction of AUC increase was performed using the in vitro data generated. 4. Interestingly, epoxide hydrolase activity was found to be much higher in human hepatocytes compared to liver microsomes/cytosol. Even though assessed on a limited number of compounds, this study demonstrated that the use of hepatocytes seems to be a more relevant model to assess and predict DDI at the epoxide hydrolase level.

  4. Inhibition of soluble epoxide hydrolase increases coronary perfusion in mice

    PubMed Central

    Qin, Jun; Sun, Dong; Jiang, Houli; Kandhi, Sharath; Froogh, Ghezal; Hwang, Sung Hee; Hammock, Bruce D; Wolin, Michael S; Thompson, Carl I; Hintze, Thomas H; Huang, An

    2015-01-01

    Roles of soluble epoxide hydrolase (sEH), the enzyme responsible for hydrolysis of epoxyeicosatrienoic acids (EETs) to their diols (DHETs), in the coronary circulation and cardiac function remain unknown. We tested the hypothesis that compromising EET hydrolysis/degradation, via sEH deficiency, lowers the coronary resistance to promote cardiac perfusion and function. Hearts were isolated from wild type (WT), sEH knockout (KO) mice and WT mice chronically treated with t-TUCB (sEH inhibitor), and perfused with constant flow at different pre-loads. Compared to WT controls, sEH-deficient hearts required significantly greater basal coronary flow to maintain the perfusion pressure at 100 mmHg and exhibited a greater reduction in vascular resistance during tension-induced heart work, implying a better coronary perfusion during cardiac performance. Cardiac contractility, characterized by developed tension in response to changes in preload, was potentially increased in sEH-KO hearts, manifested by an enlarged magnitude at each step-wise increase in end-diastolic to peak-systolic tension. 14,15-EEZE (EET antagonist) prevented the adaptation of coronary circulation in sEH null hearts whereas responses in WT hearts were sensitive to the inhibition of NO. Cardiac expression of EET synthases (CYP2J2/2C29) was comparable in both genotypic mice whereas, levels of 14,15-, 11,12- and 8,9-EETs were significantly higher in sEH-KO hearts, accompanied with lower levels of DHETs. In conclusion, the elevation of cardiac EETs, as a function of sEH deficiency, plays key roles in the adaptation of coronary flow and cardiac function. PMID:26071213

  5. Similarities between catalase and cytosolic epoxide hydrolase.

    PubMed

    Guenthner, T M; Qato, M; Whalen, R; Glomb, S

    1989-01-01

    Cytosolic epoxide hydrolase, measured as trans-stilbene oxide hydrolase activity, was isolated and purified from human and guinea pig liver cytosol. Antiserum to the guinea pig liver preparation reacted strongly with bovine liver catalase. We determined that this lack of selectivity of the antiserum was due to catalase contamination of the epoxide hydrolase preparation. We also determined that several commercial catalase preparations are contaminated with cytosolic epoxide hydrolase. Our human epoxide hydrolase preparation contained no detectable catalase contamination, yet antiserum to this protein also cross-reacted slightly with catalase, indicating some intrinsic similarity between the two enzymes. We conclude that catalase and cytosolic epoxide hydrolase contain some similar immunogenic epitopes, and we surmise that similarities between the subunits of these two enzymes may lead to their partial copurification. Functional similarities between the two enzymes are also demonstrated, as several compounds that inhibit catalase are also shown to inhibit cytosolic epoxide hydrolase activity in the same concentration range and rank order.

  6. Inhibition of soluble and microsomal epoxide hydrolase by zinc and other metals.

    PubMed

    Draper, A J; Hammock, B D

    1999-11-01

    Inhibition of xenobiotic-metabolizing enzymes by metals may represent an important mechanism in regulating enzyme activity. Fourteen cations were evaluated for inhibition of microsomal epoxide hydrolase (mEH) (mouse, rat, and human liver), soluble epoxide hydrolase (sEH) (mouse, rat, and human liver), and recombinant potato sEH. Of the metals tested, Hg2+ and Zn2+ were the strongest inhibitors of mEH, while Cd2+ and Cu2+ were also strong inhibitors of sEH (I50 for all approximately 20 microM). Nickel (divalent) and Pb2+ were moderate inhibitors, but Al2+, Ba2+, Ca2+, Co2+, Fe2+, Fe3+, Mg2+, and Mn2+ were weak inhibitors of both mEH and sEH (less than 50% inhibition by 1 mM metal). Six anions (acetate, bromide, chloride, nitrate, perchlorate, and sulfate) were tested and found to have no effect on the inhibition of sEH or mEH by cations. The kinetics and type of inhibition for zinc inhibition of sEH and mEH were examined for mouse, rat, human, and potato. Zinc inhibits mEH in a competitive manner. Inhibition of human and potato sEH was noncompetitive, but interestingly, zinc inhibition of mouse sEH was very strong and uncompetitive. Inhibition by zinc could be reversed by adding EDTA to the incubation buffer. Additionally, mouse liver microsomes and cytosol were incubated with these chelators. Following incubation at 4 degrees C, samples were dialyzed to remove chelator. Both mEH and sEH activity recovered was greater in samples treated with chelator than in control incubations. Similar treatment with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) did not affect enzyme activity recovered. During systemic inflammation, hepatic metallothionien is induced, and liver metal concentrations increase while serum metal concentrations are decreased. The inhibition of microsomal and soluble epoxide hydrolase by metals may represent a mechanism of down-regulation of enzyme activity during inflammation.

  7. Soluble epoxide hydrolase inhibition alleviates neuropathy in Akita (Ins2 Akita) mice.

    PubMed

    Wagner, Karen; Gilda, Jennifer; Yang, Jun; Wan, Debin; Morisseau, Christophe; Gomes, Aldrin V; Hammock, Bruce D

    2017-03-01

    The soluble epoxide hydrolase (sEH) is a regulatory enzyme responsible for the metabolism of bioactive lipid epoxides of both omega-6 and omega-3 long chain polyunsaturated fatty acids. These natural epoxides mediate cell signaling in several physiological functions including blocking inflammation, high blood pressure and both inflammatory and neuropathic pain. Inhibition of the sEH maintains the level of endogenous bioactive epoxy-fatty acids (EpFA) and allows them to exert their generally beneficial effects. The Akita (Ins2(Akita) or Ins2(C96Y)) mice represent a maturity-onset of diabetes of the young (MODY) model in lean, functionally unimpaired animals, with a sexually dimorphic disease phenotype. This allowed for a test of male and female mice in a battery of functional and nociceptive assays to probe the role of sEH in this system. The results demonstrate that inhibiting the sEH is analgesic in diabetic neuropathy and this occurs in a sexually dimorphic manner. Interestingly, sEH activity is also sexually dimorphic in the Akita model, and moreover correlates with disease status particularly in the hearts of male mice. In addition, in vivo levels of oxidized lipid metabolites also correlate with increased sEH expression and the pathogenesis of disease in this model. Thus, sEH is a target to effectively block diabetic neuropathic pain but also demonstrates a potential role in mitigating the progression of this disease.

  8. Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases.

    PubMed

    van der Werf, M J; Overkamp, K M; de Bont, J A

    1998-10-01

    An epoxide hydrolase from Rhodococcus erythropolis DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when R. erythropolis is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless enzyme. Maximal enzyme activity was measured at pH 7 and 50 degrees C. None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity. Limonene-1,2-epoxide hydrolase has a narrow substrate range. Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates. This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4'-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1, 10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.

  9. Inhibition of soluble epoxide hydrolase limits niacin-induced vasodilation in mice

    PubMed Central

    Inceoglu, A. B.; Clifton, H.L.; Yang, J.; Hegedus, C.; Hammock, B. D.; Schaefer, S.

    2012-01-01

    Background The use of niacin in the treatment of dyslipidemias is limited by the common side effect of cutaneous vasodilation, commonly termed flushing. Flushing is thought to be due to release of the vasodilatory prostanoids PGD2 and PGE2 from arachidonic acid metabolism through the cyclooxygenase (COX) pathway. Arachidonic acid is also metabolized by the cytochrome P450 system which is regulated, in part, by the enzyme soluble epoxide hydrolase (sEH). Methods: These experiments used an established murine model in which ear tissue perfusion was measured by laser Doppler to test the hypothesis that inhibition of sEH would limit niacin-induced flushing. Results: Niacin-induced flushing was reduced from 506 ± 126 to 213 ± 39 % in sEH knockout animals. Pharmacologic treatment with 3 structurally distinct sEH inhibitors similarly reduced flushing in a dose dependent manner, with maximal reduction to 143±15% of baseline flow using a concentration of 1 mg/kg TPAU (1-trifluoromethoxyphenyl-3-(1-acetylpiperidin-4-yl) urea). Systemically administered PGD2 caused ear vasodilation which was not changed by either pharmacologic sEH inhibition or by sEH gene deletion. Conclusions: Inhibition of sEH markedly reduces niacin-induced flushing in this model without an apparent effect on the response to PGD2. sEH inhibition may be a new therapeutic approach to limit flushing in humans. PMID:22526297

  10. Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR.

    PubMed

    Bahl, Christopher D; Hvorecny, Kelli L; Bomberger, Jennifer M; Stanton, Bruce A; Hammock, Bruce D; Morisseau, Christophe; Madden, Dean R

    2015-08-17

    Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking.

  11. Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies

    PubMed Central

    Morisseau, Christophe; Bernay, Maud; Escaich, Aurélie; Sanborn, James R.; Lango, Jozsef; Hammock, Bruce D.

    2011-01-01

    The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. Additionally, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases, such as emphysema, spontaneous abortion, eclampsia and several forms of cancer. Toward developing chemical tools to study mEH biological role, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide, but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Further, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value about 0.7. This new assay permits a significant decrease in labor and cost as well as offering the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions. PMID:21371418

  12. Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies.

    PubMed

    Morisseau, Christophe; Bernay, Maud; Escaich, Aurélie; Sanborn, James R; Lango, Jozsef; Hammock, Bruce D

    2011-07-01

    The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Pharmacological inhibition of soluble epoxide hydrolase ameliorates diet-induced metabolic syndrome in rats.

    PubMed

    Iyer, Abishek; Kauter, Kathleen; Alam, Md Ashraful; Hwang, Sung Hee; Morisseau, Christophe; Hammock, Bruce D; Brown, Lindsay

    2012-01-01

    The signs of metabolic syndrome following chronic excessive macronutrient intake include body weight gain, excess visceral adipose deposition, hyperglycaemia, glucose and insulin intolerances, hypertension, dyslipidaemia, endothelial damage, cardiovascular hypertrophy, inflammation, ventricular contractile dysfunction, fibrosis, and fatty liver disease. Recent studies show increased activity of soluble epoxide hydrolase (sEH) during obesity and metabolic dysfunction. We have tested whether sEH inhibition has therapeutic potential in a rat model of diet-induced metabolic syndrome. In these high-carbohydrate, high-fat-fed rats, chronic oral treatment with trans-4-[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid (t-AUCB), a potent sEH inhibitor, alleviated the signs of metabolic syndrome in vivo including glucose, insulin, and lipid abnormalities, changes in pancreatic structure, increased systolic blood pressure, cardiovascular structural and functional abnormalities, and structural and functional changes in the liver. The present study describes the pharmacological responses to this selective sEH inhibitor in rats with the signs of diet-induced metabolic syndrome.

  14. Pharmacological inhibition of soluble epoxide hydrolase prevents renal interstitial fibrogenesis in obstructive nephropathy

    PubMed Central

    Kim, Jinu; Yoon, Sang Pil; Toews, Myron L.; Imig, John D.; Hwang, Sung Hee; Hammock, Bruce D.

    2014-01-01

    Treating chronic kidney disease (CKD) has been challenging because of its pathogenic complexity. Epoxyeicosatrienoic acids (EETs) are cytochrome P-450-dependent derivatives of arachidonic acid with antihypertensive, anti-inflammatory, and profibrinolytic functions. We recently reported that genetic ablation of soluble epoxide hydrolase (sEH), an enzyme that converts EETs to less active dihydroxyeicosatrienoic acids, prevents renal tubulointerstitial fibrosis and inflammation in experimental mouse models of CKD. Here, we tested the hypothesis that pharmacological inhibition of sEH after unilateral ureteral obstruction (UUO) would attenuate tubulointerstitial fibrosis and inflammation in mouse kidneys and may provide a novel approach to manage the progression of CKD. Inhibition of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis, as demonstrated by reduced collagen deposition and myofibroblast formation after UUO. The inflammatory response was also attenuated, as demonstrated by decreased influx of neutrophils and macrophages and decreased expression of inflammatory cytokines keratinocyte chemoattractant, macrophage inflammatory protein-2, monocyte chemotactic protein-1, TNF-α, and ICAM-1 in kidneys after UUO. UUO upregulated transforming growth factor-β1/Smad3 signaling and induced NF-κB activation, oxidative stress, tubular injury, and apoptosis; in contrast, it downregulated antifibrotic factors, including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPAR-γ. sEH inhibition mitigated the aforementioned malevolent effects in UUO kidneys. These data demonstrate that pharmacological inhibition of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys by preventing tubular injury, downregulation of NF-κB, transforming growth factor-β1/Smad3, and inflammatory signaling pathways, and activation of PPAR isoforms. Our data suggest the potential use of sEH inhibitors in treating fibrogenesis

  15. Pharmacological inhibition of soluble epoxide hydrolase prevents renal interstitial fibrogenesis in obstructive nephropathy.

    PubMed

    Kim, Jinu; Yoon, Sang Pil; Toews, Myron L; Imig, John D; Hwang, Sung Hee; Hammock, Bruce D; Padanilam, Babu J

    2015-01-15

    Treating chronic kidney disease (CKD) has been challenging because of its pathogenic complexity. Epoxyeicosatrienoic acids (EETs) are cytochrome P-450-dependent derivatives of arachidonic acid with antihypertensive, anti-inflammatory, and profibrinolytic functions. We recently reported that genetic ablation of soluble epoxide hydrolase (sEH), an enzyme that converts EETs to less active dihydroxyeicosatrienoic acids, prevents renal tubulointerstitial fibrosis and inflammation in experimental mouse models of CKD. Here, we tested the hypothesis that pharmacological inhibition of sEH after unilateral ureteral obstruction (UUO) would attenuate tubulointerstitial fibrosis and inflammation in mouse kidneys and may provide a novel approach to manage the progression of CKD. Inhibition of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis, as demonstrated by reduced collagen deposition and myofibroblast formation after UUO. The inflammatory response was also attenuated, as demonstrated by decreased influx of neutrophils and macrophages and decreased expression of inflammatory cytokines keratinocyte chemoattractant, macrophage inflammatory protein-2, monocyte chemotactic protein-1, TNF-α, and ICAM-1 in kidneys after UUO. UUO upregulated transforming growth factor-β1/Smad3 signaling and induced NF-κB activation, oxidative stress, tubular injury, and apoptosis; in contrast, it downregulated antifibrotic factors, including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPAR-γ. sEH inhibition mitigated the aforementioned malevolent effects in UUO kidneys. These data demonstrate that pharmacological inhibition of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys by preventing tubular injury, downregulation of NF-κB, transforming growth factor-β1/Smad3, and inflammatory signaling pathways, and activation of PPAR isoforms. Our data suggest the potential use of sEH inhibitors in treating fibrogenesis

  16. Inhibition of soluble epoxide hydrolase attenuates endothelial dysfunction in animal models of diabetes, obesity and hypertension.

    PubMed

    Zhang, Le-Ning; Vincelette, Jon; Chen, Dawn; Gless, Richard D; Anandan, Sampath-Kumar; Rubanyi, Gabor M; Webb, Heather K; MacIntyre, D Euan; Wang, Yi-Xin Jim

    2011-03-01

    Endothelial dysfunction is a hallmark of, and plays a pivotal role in the pathogenesis of cardiometabolic diseases, including type II diabetes, obesity, and hypertension. It has been well established that epoxyeicosatrienoic acids (EETs) act as an endothelial derived hyperpolarization factor (EDHF). Soluble epoxide hydrolase (s-EH) rapidly hydrolyses certain epoxylipids (e.g. EETs) to less bioactive diols (DHETs), thereby attenuating the evoked vasodilator effects. The aim of the present study was to examine if inhibition of s-EH can restore impaired endothelial function in three animal models of cardiometabolic diseases. Isolated vessel rings of the aorta and/or mesenteric artery from mice or rats were pre-contracted using phenylephrine or U46619. Endothelium-dependent and independent vasorelaxation to acetylcholine and sodium nitroprusside (SNP) were measured using wire myography in vessels isolated from db/db or diet-induced obesity (DIO) mice, and angiotensin II-induced hypertensive rats treated chronically with s-EH inhibitors AR9281 or AR9276 or with vehicle. Vasorelaxation to acetylcholine, but not to SNP was severely impaired in all three animal models. Oral administration of AR9281 or AR9276 abolished whole blood s-EH activity, elevated epoxy/diol lipid ratio, and abrogated endothelial dysfunction in all three models. Incubating the mesenteric artery of db/db mice with L-NAME and indomethacin to block nitric oxide (NO) and prostacyclin formation did not affect AR9821-induced improvement of endothelial function. These data indicate that inhibition of s-EH ameliorates endothelial dysfunction and that effects in the db/db model are independent of the presence of NO and cyclooxygenase derived prostanoids. Thus, preserving vasodilator EETs by inhibition of s-EH may be of therapeutic benefit by improving endothelial function in cardiometabolic diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Inhibition of soluble epoxide hydrolase in mice promotes reverse cholesterol transport and regression of atherosclerosis.

    PubMed

    Shen, Li; Peng, Hongchun; Peng, Ran; Fan, Qingsong; Zhao, Shuiping; Xu, Danyan; Morisseau, Christophe; Chiamvimonvat, Nipavan; Hammock, Bruce D

    2015-04-01

    Adipose tissue is the body largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), which maintains plasma high-density lipoprotein (HDL) levels. HDLs have a protective role in atherosclerosis by mediating reverse cholesterol transport (RCT). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition has various beneficial effects on cardiovascular disease. The sEH is highly expressed in adipocytes, and it converts epoxyeicosatrienoic acids (EETs) into less bioactive dihydroxyeicosatrienoic acids. We previously showed that increasing EETs levels with a sEH inhibitor (sEHI) (t-AUCB) resulted in elevated ABCA1 expression and promoted ABCA1-mediated cholesterol efflux from 3T3-L1 adipocytes. The present study investigates the impacts of t-AUCB in mice deficient for the low density lipoprotein (LDL) receptor (Ldlr(-/-) mice) with established atherosclerotic plaques. The sEH inhibitor delivered in vivo for 4 weeks decreased the activity of sEH in adipose tissue, enhanced ABCA1 expression and cholesterol efflux from adipose depots, and consequently increased HDL levels. Furthermore, t-AUCB enhanced RCT to the plasma, liver, bile and feces. It also showed the reduction of plasma LDL-C levels. Consistently, t-AUCB-treated mice showed reductions in the size of atherosclerotic plaques. These studies establish that raising adipose ABCA1 expression, cholesterol efflux, and plasma HDL levels with t-AUCB treatment promotes RCT, decreasing LDL-C and atherosclerosis regression, suggesting that sEH inhibition may be a promising strategy to treat atherosclerotic vascular disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Inhibition of soluble epoxide hydrolase modulates inflammation and autophagy in obese adipose tissue and liver: Role for omega-3 epoxides

    PubMed Central

    López-Vicario, Cristina; Alcaraz-Quiles, José; García-Alonso, Verónica; Rius, Bibiana; Hwang, Sung H.; Titos, Esther; Lopategi, Aritz; Hammock, Bruce D.; Arroyo, Vicente; Clària, Joan

    2015-01-01

    Soluble epoxide hydrolase (sEH) is an emerging therapeutic target in a number of diseases that have inflammation as a common underlying cause. sEH limits tissue levels of cytochrome P450 (CYP) epoxides derived from omega-6 and omega-3 polyunsaturated fatty acids (PUFA) by converting these antiinflammatory mediators into their less active diols. Here, we explored the metabolic effects of a sEH inhibitor (t-TUCB) in fat-1 mice with transgenic expression of an omega-3 desaturase capable of enriching tissues with endogenous omega-3 PUFA. These mice exhibited increased CYP1A1, CYP2E1, and CYP2U1 expression and abundant levels of the omega-3–derived epoxides 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic (19,20-EDP) in insulin-sensitive tissues, especially liver, as determined by LC-ESI-MS/MS. In obese fat-1 mice, t-TUCB raised hepatic 17,18-EEQ and 19,20-EDP levels and reinforced the omega-3–dependent reduction observed in tissue inflammation and lipid peroxidation. t-TUCB also produced a more intense antisteatotic action in obese fat-1 mice, as revealed by magnetic resonance spectroscopy. Notably, t-TUCB skewed macrophage polarization toward an antiinflammatory M2 phenotype and expanded the interscapular brown adipose tissue volume. Moreover, t-TUCB restored hepatic levels of Atg12-Atg5 and LC3-II conjugates and reduced p62 expression, indicating up-regulation of hepatic autophagy. t-TUCB consistently reduced endoplasmic reticulum stress demonstrated by the attenuation of IRE-1α and eIF2α phosphorylation. These actions were recapitulated in vitro in palmitate-primed hepatocytes and adipocytes incubated with 19,20-EDP or 17,18-EEQ. Relatively similar but less pronounced actions were observed with the omega-6 epoxide, 14,15-EET, and nonoxidized DHA. Together, these findings identify omega-3 epoxides as important regulators of inflammation and autophagy in insulin-sensitive tissues and postulate sEH as a druggable target in metabolic

  19. Soluble Epoxide Hydrolase Inhibition and Epoxyeicosatrienoic Acid Treatment Improve Vascularization of Engineered Skin Substitutes

    PubMed Central

    Hahn, Jennifer M.; McFarland, Kevin L.; Combs, Kelly A.; Lee, Kin Sing Stephen; Inceoglu, Bora; Wan, Debin; Boyce, Steven T.; Hammock, Bruce D.

    2016-01-01

    Background: Autologous engineered skin substitutes comprised of keratinocytes, fibroblasts, and biopolymers can serve as an adjunctive treatment for excised burns. However, engineered skin lacks a vascular plexus at the time of grafting, leading to slower vascularization and reduced rates of engraftment compared with autograft. Hypothetically, vascularization of engineered skin grafts can be improved by treatment with proangiogenic agents at the time of grafting. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase (sEH). EETs have multiple biological activities and have been shown to promote angiogenesis. Inhibitors of sEH (sEHIs) represent attractive therapeutic agents because they increase endogenous EET levels. We investigated sEHI administration, alone or combined with EET treatment, for improved vascularization of engineered skin after grafting to mice. Methods: Engineered skin substitutes, prepared using primary human fibroblasts and keratinocytes, were grafted to full-thickness surgical wounds in immunodeficient mice. Mice were treated with the sEHI 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered in drinking water throughout the study period, with or without topical EET treatment, and were compared with vehicle-treated controls. Vascularization was quantified by image analysis of CD31-positive areas in tissue sections. Results: At 2 weeks after grafting, significantly increased vascularization was observed in the TPPU and TPPU + EET groups compared with controls, with no evidence of toxicity. Conclusions: The results suggest that sEH inhibition can increase vascularization of engineered skin grafts after transplantation, which may contribute to enhanced engraftment and improved treatment of full-thickness wounds. PMID:28293507

  20. Inhibition of soluble epoxide hydrolase prevents renal interstitial fibrosis and inflammation

    PubMed Central

    Kim, Jinu; Imig, John D.; Yang, Jun; Hammock, Bruce D.

    2014-01-01

    The pathophysiological events that lead to renal interstitial fibrogenesis are incompletely understood. Epoxyeicosatrienoic acid (EET), an arachidonic acid metabolite, has anti-inflammatory and profibrinolytic functions. Soluble epoxide hydrolase (sEH) converts EET to less active dihydroxyeicosatrienoic acid. Here, we tested the hypothesis that sEH deficiency would prevent tubulointerstitial fibrosis and inflammation induced by unilateral ureteral obstruction (UUO) in mouse kidneys. The loss of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis as demonstrated by reduced collagen deposition and myofibroblast formation at 3 and 10 days after UUO. The inflammatory response was prevented as demonstrated by decreased influx of neutrophil and macrophage, expression of inflammatory cytokines, and chemotactic factors in sEH-deficient UUO kidneys. Pharmacological inhibition of sEH also prevented inflammation and fibrosis after UUO. Next, we delved into the molecular mechanisms piloting the beneficial effects of sEH deficiency in renal fibrosis. UUO upregulated profibrotic factors associated with transforming growth factor (TGF)-β1/Smad3 signaling, oxidative stress, and NF-κB activation, and downregulated antifibrotic factors including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPARγ, but the loss of sEH prevented these adverse effects in UUO kidneys. Furthermore, administration of PPAR antagonists enhanced myofibroblast formation and activation of Smad3 and NF-κB p65, effects that were prevented by sEH deficiency in UUO kidneys. These data demonstrate that loss of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys via activation of PPAR isoforms and downregulation of NF-κB, TGF-β1/Smad3, and inflammatory signaling pathways. Our data suggest the potential use of sEH inhibitors in treating fibrotic diseases. PMID:25164080

  1. INHIBITION OF SOLUBLE EPOXIDE HYDROLASE DOES NOT PROTECT AGAINST ENDOTOXIN-MEDIATED HEPATIC INFLAMMATION

    PubMed Central

    Fife, Kimberly L.; Liu, YingMei; Schmelzer, Kara R.; Tsai, Hsing-Ju; Kim, In-Hae; Morisseau, Christophe; Hammock, Bruce D.; Kroetz, Deanna L.

    2009-01-01

    Epoxyeicosatrienoic acids (EETs) are derived from cytochrome P450 (CYP)-catalyzed epoxygenation of arachidonic acid and have emerged as important mediators of numerous biological effects. The major elimination pathway for EETs is through soluble epoxide hydrolase (sEH) catalyzed metabolism to dihydroxyeicosatrienoic acids (DHETs). Based on previous studies showing that EETs have anti-inflammatory effects, we hypothesized that chronic inhibition of sEH would attenuate a lipopolysaccharide (LPS)-induced inflammatory response in vivo. Continuous dosing of the sEH inhibitors 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a polyethylene glycol ester of AUDA (AUDA-PEG), and 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)pentyl)urea (AEPU) resulted in robust exposure to the inhibitor and target engagement, as evidenced by significant increases in plasma EET/DHET ratios following six days of inhibitor treatment. However, sEH inhibitor treatment was not associated with an attenuation of LPS-induced inflammatory gene expression in the liver and AUDA did not protect from LPS-induced neutrophil infiltration. Furthermore, Ephx2 −/− mice that lack sEH expression and have significantly increased plasma EET/DHET ratios were not protected from LPS-induced inflammatory gene expression or neutrophil accumulation in the liver. LPS did have an effect on sEH expression and function, as evident from a significant downregulation of Ephx2 mRNA and a significant shift in plasma EET/DHET ratios four hours after LPS treatment. In conclusion, there was no evidence that increasing EET levels in vivo could modulate an LPS-induced inflammatory response in the liver. However, LPS did have significant effects on plasma eicosanoid levels and hepatic Ephx2 expression, suggesting that in vivo EET levels are modulated in response to an inflammatory signal. PMID:18815352

  2. Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

    PubMed Central

    Olson, Lauren; Harder, Adam; Isbell, Marilyn; Imig, John D.; Gutterman, David D.; Falck, J. R.; Campbell, William B.

    2011-01-01

    Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H2O2 causes vasoconstriction. To determine the physiological contribution of H2O2, catalase is used to inactivate H2O2. However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10–50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1–10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (Vmax = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase−1·min−1, respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H2O2 and EETs. PMID:21753077

  3. Soluble epoxide hydrolase activity and pharmacologic inhibition in horses with chronic severe laminitis.

    PubMed

    Guedes, A; Galuppo, L; Hood, D; Hwang, S H; Morisseau, C; Hammock, B D

    2017-05-01

    The roles of soluble epoxide hydrolase and lipid mediators in inflammatory and neuropathic pain could be relevant in laminitis pain management. To determine soluble epoxide hydrolase (sEH) activity in the digital laminae, sEH inhibitor potency in vitro, and efficacy of a sEH inhibitor as an adjunct analgesic therapy in chronic laminitic horses. In vitro experiments and clinical case series. sEH activity was measured in digital laminae from euthanised healthy and laminitic horses (n = 5-6/group). Potency of 7 synthetic sEH inhibitors was determined in vitro using equine liver cytosol. One of them (t-TUCB; 0.1 mg/kg bwt i.v. every 24 h) was selected based on potency and stability, and used as adjunct therapy in 10 horses with severe chronic laminitis (Obel grades 2, one horse; 3-4, nine horses). Daily assessments of forelimb lifts, pain scores, physiologic and laboratory examinations were performed before (baseline) and during t-TUCB treatment. Data are presented as mean ± s.d. and 95% confidence intervals (CI). sEH activity in the digital laminae from laminitic horses (0.9±0.6 nmol/min/mg; 95% CI 0.16-1.55 nmol/min/mg) was significantly greater (P = 0.01) than in healthy horses (0.17±0.09 nmol/min/mg; CI 0.07-0.26 nmol/min/mg). t-TUCB as an adjunct analgesic up to 10 days (4.3±3 days) in laminitic horses was associated with significant reduction in forelimb lifts (36±22%; 95% CI 9-64%) and in pain scores (18±23%; 95% CI 2-35%) compared with baseline (P = 0.04). One horse developed gas colic and another corneal vascularisation in a blind eye during treatment. No other significant changes were observed. Absence of control group and evaluator blinding in case series. sEH activity is significantly higher in the digital laminae of actively laminitic compared with healthy horses, and use of a potent inhibitor of equine sEH as adjunct analgesic therapy appears to decrease signs of pathologic pain in laminitic horses. © 2016 EVJ Ltd.

  4. Pharmacological inhibition of soluble epoxide hydrolase or genetic deletion reduces diclofenac-induced gastric ulcers.

    PubMed

    Goswami, Sumanta Kumar; Rand, Amelia Ann; Wan, Debin; Yang, Jun; Inceoglu, Bora; Thomas, Melany; Morisseau, Christophe; Yang, Guang-Yu; Hammock, Bruce D

    2017-07-01

    This research was conducted to evaluate the hypothesis that gastric ulcers caused by the NSAID diclofenac sodium (DCF) can be prevented by the soluble epoxide hydrolase inhibitor TPPU. Mice were administered a single dose of 10, 30 or 100mg/kg of DCF. Once an ulcerative dose of DCF was chosen, mice were pretreated with TPPU for 7days at 0.1mg/kg to evaluate anti-ulcer effects of the sEH inhibitor on anatomy, histopathology, pH, inflammatory markers and epithelial apoptosis of stomachs. Diclofenac caused ulceration of the stomach at a dose of 100mg/kg and a time post dose of 6h. Ulcers generated under these conditions were associated with a significant increase in the levels of TNF-α and IL-6 in serum and increased apoptosis compared to control mice. Pretreatment with TPPU resulted in a decrease of ulceration in mice treated with DCF with a significant decrease in the level of apoptosis, TNF-α and IL-6 in the serum in comparison to diclofenac-treated mice. TPPU did not affect the pH of the stomach, whereas omeprazole elevated the pH of the stomach as expected. A similar anti-ulcer effect was observed in sEH gene knockout mice treated with DCF. The sEH inhibitor TPPU decreases the NSAID-induced stomach ulcers. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress

    PubMed Central

    Ren, Qian; Ma, Min; Ishima, Tamaki; Morisseau, Christophe; Yang, Jun; Wagner, Karen M.; Zhang, Ji-chun; Yang, Chun; Yao, Wei; Dong, Chao; Han, Mei; Hammock, Bruce D.; Hashimoto, Kenji

    2016-01-01

    Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression. PMID:26976569

  6. Flaxseed consumption reduces blood pressure in patients with hypertension by altering circulating oxylipins via an α-linolenic acid-induced inhibition of soluble epoxide hydrolase.

    PubMed

    Caligiuri, Stephanie P B; Aukema, Harold M; Ravandi, Amir; Guzman, Randy; Dibrov, Elena; Pierce, Grant N

    2014-07-01

    In a randomized, double-blinded, controlled clinical trial, participants with peripheral arterial disease (75% hypertensive) consumed 30 g of milled flaxseed/d for 6 months. The flaxseed group exhibited significant reductions in systolic (-10 mm Hg) and diastolic (-7 mm Hg) blood pressure. Flaxseed contains the n3 fatty acid α-linolenic acid. Plasma α-linolenic acid increased with ingestion of flaxseed and was inversely associated with blood pressure. However, the antihypertensive mechanism was unclear. Oxylipins derived from polyunsaturated fatty acids regulate vascular tone. Therefore, the objective was to examine whether flaxseed consumption altered plasma oxylipins in a manner that influenced blood pressure. Plasma of FlaxPAD (Flaxseed for Peripheral Arterial Disease) participants underwent solid phase extraction and high-performance liquid chromatography-mass spectrometry/mass spectrometry analysis. The flaxseed group exhibited significant decreases in 8 plasma oxylipins versus control. Six of these (5,6-, 8,9-, 11,12-, 14,15-dihydroxyeicosatrienoic acid and 9,10- and 12,13-dihydroxyoctadecenoic acid) were products of soluble epoxide hydrolase, a pharmacological target for antihypertensive treatment. Patients exhibiting a decrease in total plasma soluble epoxide hydrolase-derived oxylipins, exhibited a significant decrease in systolic blood pressure (mean [95% confidence interval], -7.97 [-14.4 to -1.50] mm Hg) versus those who exhibited increased plasma soluble epoxide hydrolase-derived oxylipins (+3.17 [-4.78 to 11.13] mm Hg). These data suggest that a flaxseed bioactive may have decreased blood pressure via soluble epoxide hydrolase inhibition. Using a soluble epoxide hydrolase inhibitor screening assay, increasing concentrations of α-linolenic acid decreased soluble epoxide hydrolase activity (P=0.0048; ρ=-0.94). In conclusion, α-linolenic acid in flaxseed may have inhibited soluble epoxide hydrolase, which altered oxylipin concentrations that

  7. 3-D QSAR ANALYSIS OF INHIBITION OF MURINE SOLUBLE EPOXIDE HYDROLASE (MSEH) BY BENZOYLUREAS, ARYLUREAS, AND THEIR ANALOGUES. (R825433)

    EPA Science Inventory

    Two hundred and seventy-one compounds including benzoylureas, arylureas and related compounds were assayed using recombinant murine soluble epoxide hydrolase (MsEH) produced from a baculovirus expression system. Among all the insect growth regulators assayed, 18 benzoylphenylu...

  8. 3-D QSAR ANALYSIS OF INHIBITION OF MURINE SOLUBLE EPOXIDE HYDROLASE (MSEH) BY BENZOYLUREAS, ARYLUREAS, AND THEIR ANALOGUES. (R825433)

    EPA Science Inventory

    Two hundred and seventy-one compounds including benzoylureas, arylureas and related compounds were assayed using recombinant murine soluble epoxide hydrolase (MsEH) produced from a baculovirus expression system. Among all the insect growth regulators assayed, 18 benzoylphenylu...

  9. Soluble Epoxide Hydrolase Deficiency Inhibits Dextran Sulfate Sodium-induced Colitis and Carcinogenesis in Mice

    PubMed Central

    DONG, HUA; LIAO, JIE; HAMMOCK, BRUCE D.; YANG, GUANG-YU

    2014-01-01

    Soluble epoxide hydrolase (sEH) hydrolyses/inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs) to their corresponding diols, and targeting sEH leads to strong anti-inflammatory effects. In the present study, using a tissue microarray and immunohistochemical approach, a significant increase of sEH expression was identified in ulcerative colitis (UC)-associated dysplasia and adenocarcinoma. The effects of deficiency in the sEH gene were determined on dextran sulfate sodium (DSS) colitis-induced carcinogenesis. The effects of EETs on lipopolysaccharide (LPS)-activated macrophages were analyzed in vitro. With extensive histopathological and immunohistochemical analyses, compared to wild-type mice, sEH−/− mice exhibited a significant decrease in tumor incidence (13/20 vs. 6/19, p<0.05) and a markedly reduced average tumor size (59.62±20.91 mm3 vs. 22.42±11.22 mm3), and a significant number of pre-cancerous dysplasia (3±1.18 vs. 2±0.83, p<0.01). The inflammatory activity, as measured by the extent/proportion of erosion/ulceration/dense lymphoplasmacytosis (called active colitis index) in the colon, was significantly lower in sEH−/− mice (44.7%±24.9% vs. 20.2%±16.2%, p<0.01). The quantitative polymerase chain reaction (qPCR) assays demonstrated significantly low levels of cytokines/chemokines including monocyte chemoattractant protein (MCP-1), inducible nitric oxide synthase (iNOS), vasopressin-activated calcium-mobilizing (VCAM-1), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). In vitro, LPS-activated macrophages treated with 14,15-EET showed a significant reduction of LPS-triggered IL-1β and TNF-α expression. Eicosanoic acid metabolic profiling revealed a significant increase of the ratios of EETs/dihydroeicosatrienoic acids (DHETs) and epoxyoctadecennoic acid/dihydroxyoctadecenoic acid (EpOMEs/DiHOMEs). These results indicate that sEH plays an important role in the development of colitis and in inducing carcinogenesis

  10. Soluble epoxide hydrolase inhibition reveals novel biological functions of epoxyeicosatrienoic acids (EETs)

    PubMed Central

    Inceoglu, Bora; Schmelzer, Kara R.; Morisseau, Christophe; Jinks, Steve L.; Hammock, Bruce D.

    2007-01-01

    Early on, intriguing biological activities were found associated with the EETs using in vitro systems. Although the EETs other than the 5,6-isomer, are quite stable chemically, they are quickly degraded enzymatically with the sEH accounting in many cases for much of the metabolism. This rapid degradation often made it difficult to associate biological effects with the administration of EETs and other lipid epoxides particularly in vivo. Thus, it is the power to inhibit the sEH that has facilitated the demonstration of many physiological processes associated with EETs and possibly other epoxy fatty acids. In the last few years it has become clear that major roles of the EETs include modulation of blood pressure and modulation of inflammatory cascades. There are a number of other physiological functions now associated with the EETs including angiogenesis, neurohormone release, cell proliferation, G protein signaling, modulation of ion channel activity, and a variety of effects associated with modulation of NFκB. More recently we observed a role of the EETs as modulated by sEHI in reducing non-neuropathic pain. The array of biological effects observed with sEHI illustrates the power of modulating the degradation of chemical mediators in addition to the modulation of their biosynthesis, receptor binding and signal transduction. Many of these biological effects can be modulated by sEHIs but also by the natural eicosanoids and their mimics all of which offer therapeutic potential. PMID:17164131

  11. Soluble epoxide hydrolase inhibition does not prevent cardiac remodeling and dysfunction after aortic constriction in rats and mice.

    PubMed

    Morgan, Lisa A; Olzinski, Alan R; Upson, John J; Zhao, Shufang; Wang, Tao; Eisennagel, Stephen H; Hoang, Bao; Tunstead, James R; Marino, Joseph P; Willette, Robert N; Jucker, Beat M; Behm, David J

    2013-04-01

    Epoxyeicosatrienoic acids, substrates for soluble epoxide hydrolase (sEH), exhibit vasodilatory and antihypertrophic activities. Inhibitors of sEH might therefore hold promise as heart failure therapeutics. We examined the ability of sEH inhibitors GSK2188931 and GSK2256294 to modulate cardiac hypertrophy, fibrosis, and function after transverse aortic constriction (TAC) in rats and mice. GSK2188931 administration was initiated in rats 1 day before TAC, whereas GSK2256294 treatment was initiated in mice 2 weeks after TAC. Four weeks later, cardiovascular function was assessed, plasma was collected for drug and sEH biomarker concentrations, and left ventricle was isolated for messenger RNA and histological analyses. In rats, although GSK2188931 prevented TAC-mediated increases in certain genes associated with hypertrophy and fibrosis (α-skeletal actin and connective tissue growth factor), the compound failed to attenuate TAC-induced increases in left ventricle mass, posterior wall thickness, end-diastolic volume and pressure, and perivascular fibrosis. Similarly, in mice, GSK2256294 did not reverse cardiac remodeling or systolic dysfunction induced by TAC. Both compounds increased the sEH substrate/product (leukotoxin/leukotoxin diol) ratio, indicating sEH inhibition. In summary, sEH inhibition does not prevent cardiac remodeling or dysfunction after TAC. Thus, targeting sEH seems to be insufficient for reducing pressure overload hypertrophy.

  12. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    PubMed Central

    Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

    2015-01-01

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl4)-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl4-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl4-treated group relative to the control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl4-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl4, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. PMID:25827057

  13. Inhibition of soluble epoxide hydrolase contributes to the anti-inflammatory effect of antimicrobial triclocarban in a murine model

    PubMed Central

    Liu, Jun-Yan; Qiu, Hong; Morisseau, Christophe; Hwang, Sung Hee; Tsai, Hsing-Ju; Ulu, Arzu; Chiamvimonvat, Nipavan; Hammock, Bruce D

    2011-01-01

    The increasing use of the anti-microbial triclocarban (TCC) in personal care products (PCPs) has resulted in concern regarding environmental pollution. TCC is a potent inhibitor of soluble epoxide hydrolase (sEH). Inhibitors of sEH (sEHIs) are anti-inflammatory, anti-hypertensive and cardio-protective in multiple animal models. However, the in vivo effects anticipated from a sEHI have not been reported for TCC. Here we demonstrated the anti-inflammatory effects in vivo of TCC in a murine model. TCC was employed in a lipopolysaccharide (LPS)-challenged murine model. Systolic blood pressure, plasma levels of several inflammatory cytokines and chemokine, and metabolomic profile of plasma oxylipins were determined. TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. TCC significantly repressed the increased release of inflammatory cytokines and chemokine caused by LPS. Furthermore, TCC significantly shifted the oxylipin profile in vivo in a time-dependent manner towards resolution of inflammation as expected from a sEHI. These results demonstrated that at the doses used TCC is anti-inflammatory in the murine model. This study suggests that TCC may provide some benefits in humans in addition to its antimicrobial activities due to its potent inhibition of sEH. It may be a promising starting point for developing new low volume high value applications of TCC. However these biological effects also caution against the general over use of TCC in PCPs. PMID:21741984

  14. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    SciTech Connect

    Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

    2015-07-15

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.

  15. Epoxy Fatty Acids and Inhibition of the Soluble Epoxide Hydrolase Selectively Modulate GABA Mediated Neurotransmission to Delay Onset of Seizures

    PubMed Central

    Inceoglu, Bora; Zolkowska, Dorota; Yoo, Hyun Ju; Wagner, Karen M.; Yang, Jun; Hackett, Edward; Hwang, Sung Hee; Lee, Kin Sing Stephen; Rogawski, Michael A.; Morisseau, Christophe; Hammock, Bruce D.

    2013-01-01

    In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders. PMID:24349022

  16. Inhibition of soluble epoxide hydrolase contributes to the anti-inflammatory effect of antimicrobial triclocarban in a murine model

    SciTech Connect

    Liu Junyan; Qiu Hong; Morisseau, Christophe; Hwang, Sung Hee; Tsai, Hsing-Ju; Ulu, Arzu; Chiamvimonvat, Nipavan; Hammock, Bruce D.

    2011-09-01

    The increasing use of the antimicrobial triclocarban (TCC) in personal care products (PCPs) has resulted in concern regarding environmental pollution. TCC is a potent inhibitor of soluble epoxide hydrolase (sEH). Inhibitors of sEH (sEHIs) are anti-inflammatory, anti-hypertensive and cardio-protective in multiple animal models. However, the in vivo effects anticipated from a sEHI have not been reported for TCC. Here we demonstrated the anti-inflammatory effects in vivo of TCC in a murine model. TCC was employed in a lipopolysaccharide (LPS)-challenged murine model. Systolic blood pressure, plasma levels of several inflammatory cytokines and chemokine, and metabolomic profile of plasma oxylipins were determined. TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. TCC significantly repressed the increased release of inflammatory cytokines and chemokine caused by LPS. Furthermore, TCC significantly shifted the oxylipin profile in vivo in a time-dependent manner towards resolution of inflammation as expected from a sEHI. These results demonstrated that at the doses used TCC is anti-inflammatory in the murine model. This study suggests that TCC may provide some benefits in humans in addition to its antimicrobial activities due to its potent inhibition of sEH. It may be a promising starting point for developing new low volume high value applications of TCC. However these biological effects also caution against the general over use of TCC in PCPs. - Graphical abstract: Display Omitted Research Highlights: > Anti-microbial triclocarban (TCC) is anti-inflammatory in a murine model. > TCC significantly shifted the oxylipin profile in vivo as expected from a sEHI. > TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. > TCC significantly repressed LPS-induced increased release of inflammatory cytokines.

  17. Hydrolysis of the 2',3'-allylic epoxides of allylbenzene, estragole, eugenol, and safrole by both microsomal and cytosolic epoxide hydrolases.

    PubMed

    Luo, G; Qato, M K; Guenthner, T M

    1992-01-01

    2',3'-Allylic epoxide derivatives of allylbenzene and its analogs estragole, eugenol, and safrole were synthesized, and their enzymatic conversion to dihydrodiols by cytosolic and microsomal epoxide hydrolases was examined. All four epoxides were good substrates for both epoxide hydrolases, with Michaelis constants in the low micromolar range. Two putatively selective inhibitors of cytosolic and microsomal epoxide hydrolases, trichloropropylene oxide and nordihydroguaiaretic acid, were used to inhibit the hydrolysis of these allylic epoxides. Minimal selectivity toward either hydrolase was seen with either inhibitor, suggesting that the "selectivity" of these inhibitors is highly substrate-dependent. The susceptibilities of these epoxides to rapid hydrolysis by both epoxide hydrolases may explain their low genotoxic potencies in vivo.

  18. Inhibition of soluble epoxide hydrolase reduces LPS-induced thermal hyperalgesia and mechanical allodynia in a rat model of inflammatory pain

    PubMed Central

    Inceoglu, Bora; Jinks, Steven L.; Schmelzer, Kara R.; Waite, Troy; Kim, In Hae; Hammock, Bruce D.

    2007-01-01

    Soluble epoxide hydrolases catalyze the hydrolysis of epoxides in acyclic systems. In man this enzyme is the product of a single copy gene (EPXH-2) present on chromosome 8. The human sEH is of interest due to emerging roles of its endogenous substrates, epoxygenated fatty acids, in inflammation and hypertension. One of the consequences of inhibiting sEH in rodent inflammation models is a profound decrease in the production of pro-inflammatory and proalgesic lipid metabolites including prostaglandins. This prompted us to hypothesize that sEH inhibitors may have antinociceptive properties. Here we tested if sEH inhibitors can reduce inflammatory pain. Hyperalgesia was induced by intraplantar LPS injection and sEH inhibitors were delivered topically. We found that two structurally dissimilar but equally potent sEH inhibitors can be delivered through the transdermal route and that sEH inhibitors effectively attenuate thermal hyperalgesia and mechanical allodynia in rats treated with LPS. In addition we show that epoxydized arachidonic acid metabolites, EETs, are also effective in attenuating thermal hyperalgesia in this model. In parallel with the observed biological activity metabolic analysis of oxylipids showed that inhibition of sEH resulted with a decrease in PGD2 levels and sEH generated degradation products of linoleic and arachidonic acid metabolites with a concomitant increase in epoxides of linoleic acid. These data show that inhibition of sEH may become a viable therapeutic strategy to attain analgesia. PMID:16962614

  19. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    PubMed Central

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

  20. Inhibition of soluble epoxide hydrolase counteracts the development of renal dysfunction and progression of congestive heart failure in Ren-2 transgenic hypertensive rats with aorto-caval fistula.

    PubMed

    Červenka, Luděk; Melenovský, Vojtěch; Husková, Zuzana; Škaroupková, Petra; Nishiyama, Akira; Sadowski, Janusz

    2015-07-01

    The detailed mechanisms determining the course of congestive heart failure (CHF) in hypertensive subjects with associated renal dysfunction remain unclear. In Ren-2 transgenic rats (TGR), a model of angiotensin II (ANG II)-dependent hypertension, CHF was induced by volume overload achieved by creation of the aorto-caval fistula (ACF). In these rats we investigated the putative pathophysiological contribution of epoxyeicosatrienoic acids (EETs) and compared it with the role of the renin-angiotensin system (RAS). We found that untreated ACF TGR exhibited marked intrarenal and myocardial deficiency of EETs and impairment of renal function. Chronic treatment of these rats with cis-4-[4-(3-adamantan-1-yl-ureido)cyclohexyloxy]benzoic acid (c-AUCB, 3 mg/L in drinking water), an inhibitor of soluble epoxide hydrolase (sEH) which normally degrades EETs, increased intrarenal and myocardial EETs, markedly improved survival rate, and increased renal blood flow, glomerular filtration rate and fractional sodium excretion, without altering RAS activity. Chronic angiotensin-converting enzyme inhibition (ACEi) with trandolapril, (6 mg/L in drinking water) improved survival rate even more, and also inhibited the development of renal dysfunction; these beneficial actions were associated with significant suppression of the vasoconstrictor/sodium retaining axis and further activation of the vasodilatory/natriuretic axis of the systemic and intrarenal RAS, without modifying tissue availability of biologically active fatty acid epoxides. In conclusion, these findings strongly suggest that chronic sEH inhibition and chronic treatment with ACEi, each of them altering a different vasoactive system, delay or even prevent the onset of decompensation of CHF in ACF TGR, probably by preventing the development of renal dysfunction.

  1. Characterization of multiple epoxide hydrolase activities in mouse liver nuclear envelope.

    PubMed

    Guenthner, T M

    1986-10-01

    A nuclear envelope-associated epoxide hydrolase in mouse liver that hydrates trans-stilbene oxide has been identified and characterized. This epoxide hydrolase is distinct from the enzyme in nuclear envelopes that hydrates benzo[a]pyrene 4,5-oxide and other arene oxides. This distinction was demonstrated by the criteria of pH optima, response to specific inhibitors in vitro, and precipitation by specific antibodies. The new epoxide hydrolase had a pH optimum of 6.8, was poorly inhibited by trichloropropene oxide, was potently inhibited by 4-phenylchalcone oxide, and did not bind to antiserum against benzo[a]pyrene 4,5-oxide hydrolase. This nuclear enzyme is similar in many of its properties to cytosolic and microsomal trans-stilbene oxide hydrolases and may be nuclear envelope-bound form of these other epoxide hydrolases. It differed from these other trans-stilbene oxide hydrolases in that its affinities for both trans-stilbene oxide (measured as apparent Km) and 4-phenylchalcone oxide (measured as I50) were 4- to 20-fold lower than those of either the cytosolic or microsomal forms.

  2. Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

    SciTech Connect

    Vito, Stephen T.; Austin, Adam T.; Banks, Christopher N.; Inceoglu, Bora; Bruun, Donald A.; Zolkowska, Dorota; Tancredi, Daniel J.; Rogawski, Michael A.; Hammock, Bruce D.; Lein, Pamela J.

    2014-12-01

    Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABA{sub A}R) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABA{sub A}R positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15 mg/kg, ip). Administration of a high dose of diazepam (5 mg/kg, ip) immediately following the second clonic seizure (approximately 20 min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABA{sub A}R antagonists. The sEH inhibitor TUPS (1 mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5 mg/kg, ip) and TUPS (1 mg/kg, ip, starting 1 h after diazepam and repeated every 24 h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. - Highlights: • Acute TETS intoxication causes delayed and persistent neuroinflammation. • Diazepam given post-TETS prevents lethal tonic seizures but not neuroinflammation. • A soluble epoxide hydrolase

  3. Inhibition of soluble epoxide hydrolase after cardiac arrest/cardiopulmonary resuscitation induces a neuroprotective phenotype in activated microglia and improves neuronal survival.

    PubMed

    Wang, Jianming; Fujiyoshi, Tetsuhiro; Kosaka, Yasuharu; Raybuck, Jonathan D; Lattal, K Matthew; Ikeda, Mizuko; Herson, Paco S; Koerner, Ines P

    2013-10-01

    Cardiac arrest (CA) causes hippocampal neuronal death that frequently leads to severe loss of memory function in survivors. No specific treatment is available to reduce neuronal death and improve functional outcome. The brain's inflammatory response to ischemia can exacerbate injury and provides a potential treatment target. We hypothesized that microglia are activated by CA and contribute to neuronal loss. We used a mouse model to determine whether pharmacologic inhibition of the proinflammatory microglial enzyme soluble epoxide hydrolase (sEH) after CA alters microglial activation and neuronal death. The sEH inhibitor 4-phenylchalcone oxide (4-PCO) was administered after successful cardiopulmonary resuscitation (CPR). The 4-PCO treatment significantly reduced neuronal death and improved memory function after CA/CPR. We found early activation of microglia and increased expression of inflammatory tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the hippocampus after CA/CPR, which was unchanged after 4-PCO treatment, while expression of antiinflammatory IL-10 increased significantly. We conclude that sEH inhibition after CA/CPR can alter the transcription profile in activated microglia to selectively induce antiinflammatory and neuroprotective IL-10 and reduce subsequent neuronal death. Switching microglial gene expression toward a neuroprotective phenotype is a promising new therapeutic approach for ischemic brain injury.

  4. Inhibition of mutant KrasG12D-initiated murine pancreatic carcinoma growth by a dual c-Raf and soluble epoxide hydrolase inhibitor t-CUPM.

    PubMed

    Liao, Jie; Hwang, Sung Hee; Li, Haonan; Yang, Yihe; Yang, Jun; Wecksler, Aaron T; Liu, Jun-Yan; Hammock, Bruce D; Yang, Guang-Yu

    2016-02-28

    Mutant Kras and chronic pancreatitis are the most common pathological events involved in human pancreatic cancer. It has been demonstrated that c-Raf is responsible for transmitting signals from mutant Ras to its downstream signals including MEK-ERK and for initiating carcinogenesis. The soluble epoxide hydrolase (sEH), a pro-inflammatory enzyme, generally inactivates anti-inflammatory and anti-pain epoxyeicosatrienoic acids (EETs). Herein, we have synthesized a novel compound of trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM) via modifying the central phenyl ring of sorafenib and confirmed its dual inhibition of sEH and c-Raf by recombinant kinase activity assay. Pharmacokinetic analysis revealed that oral dosing of t-CUPM resulted in higher blood levels than that of sorafenib throughout the complete time course (48 h). The effect of t-CUPM on the inhibition of mutant Kras(G12D)-initiated murine pancreatic cancer cell growth was determined using the mouse pancreatic carcinoma cell model obtained from LSL-Kras(G12D)/Pdx1-Cre mice and showed that t-CUPM significantly inhibited this murine pancreatic carcinoma cell growth both in vitro and in mice in vivo. Inhibition of mutant Kras-transmitted phosphorylations of cRAF/MEK/ERK was demonstrated in these pancreatic cancer cells using Western blot assay and immunohistochemical approach. Modulation of oxylipin profile, particularly increased EETs/DHET ratio by sEH inhibition, was observed in mice treated with t-CUPM. These results indicate that t-CUPM is a highly potential agent to treat pancreatic cancer via simultaneously targeting c-Raf and sEH.

  5. Soluble epoxide hydrolase inhibition improves coronary endothelial function and prevents the development of cardiac alterations in obese insulin-resistant mice

    PubMed Central

    Roche, Clothilde; Besnier, Marie; Cassel, Roméo; Harouki, Najah; Coquerel, David; Guerrot, Dominique; Nicol, Lionel; Loizon, Emmanuelle; Remy-Jouet, Isabelle; Morisseau, Christophe; Mulder, Paul; Ouvrard-Pascaud, Antoine; Madec, Anne-Marie; Richard, Vincent

    2015-01-01

    This study addressed the hypothesis that inhibiting the soluble epoxide hydrolase (sEH)-mediated degradation of epoxy-fatty acids, notably epoxyeicosatrienoic acids, has an additional impact against cardiovascular damage in insulin resistance, beyond its previously demonstrated beneficial effect on glucose homeostasis. The cardiovascular and metabolic effects of the sEH inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 10 mg/l in drinking water) were compared with those of the sulfonylurea glibenclamide (80 mg/l), both administered for 8 wk in FVB mice subjected to a high-fat diet (HFD; 60% fat) for 16 wk. Mice on control chow diet (10% fat) and nontreated HFD mice served as controls. Glibenclamide and t-AUCB similarly prevented the increased fasting glycemia in HFD mice, but only t-AUCB improved glucose tolerance and decreased gluconeogenesis, without modifying weight gain. Moreover, t-AUCB reduced adipose tissue inflammation, plasma free fatty acids, and LDL cholesterol and prevented hepatic steatosis. Furthermore, only the sEH inhibitor improved endothelium-dependent relaxations to acetylcholine, assessed by myography in isolated coronary arteries. This improvement was related to a restoration of epoxyeicosatrienoic acid and nitric oxide pathways, as shown by the increased inhibitory effects of the nitric oxide synthase and cytochrome P-450 epoxygenase inhibitors l-NA and MSPPOH on these relaxations. Moreover, t-AUCB decreased cardiac hypertrophy, fibrosis, and inflammation and improved diastolic function, as demonstrated by the increased E/A ratio (echocardiography) and decreased slope of the end-diastolic pressure-volume relation (invasive hemodynamics). These results demonstrate that sEH inhibition improves coronary endothelial function and prevents cardiac remodeling and diastolic dysfunction in obese insulin-resistant mice. PMID:25724490

  6. Post-Exposure Administration of Diazepam Combined with Soluble Epoxide Hydrolase Inhibition Stops Seizures and Modulates Neuroinflammation in a Murine Model of Acute TETS Intoxication

    PubMed Central

    Vito, Stephen T.; Austin, Adam T.; Banks, Christopher N.; Inceoglu, Bora; Bruun, Donald A.; Zolkowska, Dorota; Tancredi, Daniel J.; Rogawski, Michael A.; Hammock, Bruce D.; Lein, Pamela J.

    2014-01-01

    Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15 mg/kg, ip). Administration of a high dose of diazepam (5 mg/kg, ip) immediately following the second clonic seizure (approximately 20 min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABAAR antagonists. The sEH inhibitor TUPS (1 mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5 mg/kg, ip) and TUPS (1 mg/kg, ip, starting 1 h after diazepam and repeated every 24 h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. PMID:25448683

  7. Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication.

    PubMed

    Vito, Stephen T; Austin, Adam T; Banks, Christopher N; Inceoglu, Bora; Bruun, Donald A; Zolkowska, Dorota; Tancredi, Daniel J; Rogawski, Michael A; Hammock, Bruce D; Lein, Pamela J

    2014-12-01

    Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15mg/kg, ip). Administration of a high dose of diazepam (5mg/kg, ip) immediately following the second clonic seizure (approximately 20min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABAAR antagonists. The sEH inhibitor TUPS (1mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5mg/kg, ip) and TUPS (1mg/kg, ip, starting 1h after diazepam and repeated every 24h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Potent Urea and Carbamate Inhibitors of Soluble Epoxide Hydrolases

    NASA Astrophysics Data System (ADS)

    Morisseau, Christophe; Goodrow, Marvin H.; Dowdy, Deanna; Zheng, Jiang; Greene, Jessica F.; Sanborn, James R.; Hammock, Bruce D.

    1999-08-01

    The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inflammation mediators as well as xenobiotic transformations. Herein, we report the discovery of substituted ureas and carbamates as potent inhibitors of sEH. Some of these selective, competitive tightbinding inhibitors with nanomolar Ki values interacted stoichiometrically with the homogenous recombinant murine and human sEHs. These inhibitors enhance cytotoxicity of trans-stilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase to its toxic diol. They also reduce toxicity of leukotoxin in vivo in mice and prevent symptoms suggestive of acute respiratory distress syndrome. These potent inhibitors may be valuable tools for testing hypotheses of involvement of diol and epoxide lipids in chemical mediation in vitro or in vivo systems.

  9. Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis†

    PubMed Central

    van Loo, Bert; Kingma, Jaap; Arand, Michael; Wubbolts, Marcel G.; Janssen, Dick B.

    2006-01-01

    Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative α/β-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts. PMID:16597997

  10. Soluble epoxide hydrolase inhibition and peroxisome proliferator activated receptor γ agonist improve vascular function and decrease renal injury in hypertensive obese rats.

    PubMed

    Imig, John D; Walsh, Katie A; Hye Khan, Md Abdul; Nagasawa, Tasuku; Cherian-Shaw, Mary; Shaw, Sean M; Hammock, Bruce D

    2012-12-01

    Cardiometabolic syndrome occurs with obesity and consists of pathophysiological factors that increase the risk for cardiovascular events. Soluble epoxide hydrolase inhibition (sEHi) is a novel therapeutic approach that exerts renal and cardiovascular protection. Although sEHi as a therapeutic approach is promising, it could be more effective for the treatment of cardiometabolic syndrome when combined with peroxisome proliferator activated receptor γ (PPARγ) agonists. We hypothesized that the PPARγ agonist, rosiglitazone in combination with a sEHi (tAUCB) will provide synergistic actions to decrease blood pressure, improve vascular function, decrease inflammation, and prevent renal damage in spontaneously hypertensive obese rats (SHROB). SHROB were treated with rosiglitazone, tAUCB or the combination of tAUCB and rosiglitazone for four-weeks and compared with spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Blood pressure increased in SHROB (164 ± 7 mmHg) and decreased 10 mmHg when treated with rosiglitazone, tAUCB, or tAUCB and rosiglitazone. Mesenteric artery dilation to the K(ATP) channel opener pinacidil was attenuated in SHROB (E(Max) = 77 ± 7%), compared with WKY (E(Max) = 115 ± 19) and SHR (E(Max) = 93 ± 12%). Vasodilation to pinacidil was improved by rosiglitazone (E(Max) = 92 ± 14%) but not tAUCB. Renal macrophage infiltration increased in SHROB and significantly decreased with rosiglitazone or tAUCB and rosiglitazone treatment. Albuminuria was increased in SHROB (90 ± 20 mg/d) and was significantly decreased by the combination of tAUCB and rosiglitazone (37 ± 9 mg/d). Glomerular injury in SHROB was also significantly decreased by tAUCB and rosiglitazone. These results indicate that even though sEHi or PPARγ agonist have benefits when used individually, the combination is more beneficial for the multidisease features in cardiometabolic syndrome.

  11. Soluble epoxide hydrolase inhibition and peroxisome proliferator activated receptor γ agonist improve vascular function and decrease renal injury in hypertensive obese rats

    PubMed Central

    Imig, John D; Walsh, Katie A; Khan, Md Abdul Hye; Nagasawa, Tasuku; Cherian-Shaw, Mary; Shaw, Sean M; Hammock, Bruce D

    2013-01-01

    Cardiometabolic syndrome occurs with obesity and consists of pathophysiological factors that increase the risk for cardiovascular events. Soluble epoxide hydrolase inhibition (sEHi) is a novel therapeutic approach that exerts renal and cardiovascular protection. Although sEHi as a therapeutic approach is promising, it could be more effective for the treatment of cardiometabolic syndrome when combined with peroxisome proliferator activated receptor γ (PPARγ) agonists. We hypothesized that the PPARγ agonist, rosiglitazone in combination with a sEHi (tAUCB) will provide synergistic actions to decrease blood pressure, improve vascular function, decrease inflammation, and prevent renal damage in spontaneously hypertensive obese rats (SHROB). SHROB were treated with rosiglitazone, tAUCB or the combination of tAUCB and rosiglitazone for four-weeks and compared with spontaneously hypertensive (SHR) and Wistar–Kyoto (WKY) rats. Blood pressure increased in SHROB (164 ±7 mmHg) and decreased 10 mmHg when treated with rosiglitazone, tAUCB, or tAUCB and rosiglitazone. Mesenteric artery dilation to the KATP channel opener pinacidil was attenuated in SHROB (EMax = 77 ±7%), compared with WKY (EMax = 115 ±19) and SHR (EMax = 93 ±12%). Vasodilation to pinacidil was improved by rosiglitazone (EMax = 92 ±14%) but not tAUCB. Renal macrophage infiltration increased in SHROB and significantly decreased with rosiglitazone or tAUCB and rosiglitazone treatment. Albuminuria was increased in SHROB (90 ±20 mg/d) and was significantly decreased by the combination of tAUCB and rosiglitazone (37 ±9 mg/d). Glomerular injury in SHROB was also significantly decreased by tAUCB and rosiglitazone. These results indicate that even though sEHi or PPARγ agonist have benefits when used individually, the combination is more beneficial for the multidisease features in cardiometabolic syndrome. PMID:23354399

  12. Immunohistochemical study of epoxide hydrolase induced by trichloroethylene in rat liver

    SciTech Connect

    Kawamoto, T.; Hobara, T.; Ogino, K.; Takemoto, T.; Nakamura, K.; Imamura, A.; Koshiro, A.; Kobayashi, H.; Iwamoto, S.; Sakai, T.

    1987-10-01

    Epoxide hydrolase catalyzes the hydrolation of potentially toxic, electrophilic epoxides that are often generated during cytochrome P-450 catalyzed monooxigenation, forming the corresponding transdihydrodiols. It is well-known that trichloroethylene is metabolized by cytochrome P-450 containing mixed-function oxidase systems to trichloroethylene oxide, which decomposes to other metabolites. As trichloroethylene is an epoxide, epoxide hydrolase is suspected to catalyze the hydrolation of trichloroethylene oxide. No reports have appeared about the relationship between trichloroethylene and epoxide hydrolase. In this report, the authors studied the effect of trichloroethylene on epoxide hydrolase immunohistochemically.

  13. DEVELOPMENT OF METABOLICALLY STABLE INHIBITORS OF MAMMALIAN MICROSOMAL EPOXIDE HYDROLASE

    USDA-ARS?s Scientific Manuscript database

    The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia ...

  14. Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives.

    PubMed

    Kotik, Michael; Brichac, Jiri; Kyslík, Pavel

    2005-12-06

    Microbial isolates from biofilters and petroleum-polluted bioremediation sites were screened for the presence of enantioselective epoxide hydrolases active towards tert-butyl glycidyl ether, benzyl glycidyl ether, and allyl glycidyl ether. Out of 270 isolated strains, which comprised bacteria, yeasts, and filamentous fungi, four were selected based on the enantioselectivities of their epoxide hydrolases determined in biotransformation reactions. The enzyme of Aspergillus niger M200 preferentially hydrolyses (S)-tert-butyl glycidyl ether to (S)-3-tert-butoxy-1,2-propanediol with a relatively high enantioselectivity (the enantiomeric ratio E is about 30 at a reaction temperature of 28 degrees C). Epoxide hydrolases of Rhodotorula mucilaginosa M002 and Rhodococcus fascians M022 hydrolyse benzyl glycidyl ether with relatively low enantioselectivities, the former reacting predominantly with the (S)-enantiomer, the latter preferring the (R)-enantiomer. Enzymatic hydrolysis of allyl glycidyl ether by Cryptococcus laurentii M001 proceeds with low enantioselectivity (E=3). (R)-tert-Butyl glycidyl ether with an enantiomeric excess (ee) of over 99%, and (S)-3-tert-butoxy-1,2-propanediol with an ee-value of 86% have been prepared on a gram-scale using whole cells of A. niger M200. An enantiomeric ratio of approximately 100 has been determined under optimised biotransformation conditions with the partially purified epoxide hydrolase from A. niger M200. The regioselectivity of this enzyme was determined to be total for both (S)-tert-butyl glycidyl ether and (R)-tert-butyl glycidyl ether.

  15. Epoxide hydrolase activities and epoxy fatty acids in the mosquito Culex quinquefasciatus

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Yang, Jun; Mamatha, Dadala M.

    2015-01-01

    Culex mosquitoes have emerged as important model organisms for mosquito biology, and are disease vectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxide hydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxide hydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and other eicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be synthesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both in larvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding, and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETs can be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). The sEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can be useful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism and detoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, such as EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by female mosquitoes. PMID:25686802

  16. Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse.

    PubMed

    Guenthner, T M; Hammock, B D; Vogel, U; Oesch, F

    1981-04-10

    Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.

  17. Soluble epoxide hydrolase: Gene structure, expression and deletion

    PubMed Central

    Harris, Todd R.; Hammock, Bruce D.

    2013-01-01

    Mammalian soluble epoxide hydrolase (sEH) converts epoxides to their corresponding diols through the addition of a water molecule. sEH readily hydrolyzes lipid signaling molecules, including the epoxyeicosatrienoic acids (EETs), epoxidized lipids produced from arachidonic acid by the action of cytochrome p450s. Through its metabolism of the EETs and other lipid mediators, sEH contributes to the regulation of vascular tone, nociception, angiogenesis and the inflammatory response. Because of its central physiological role in disease states such as cardiac hypertrophy, diabetes, hypertension, and pain sEH is being investigated as a therapeutic target. This review begins with a brief introduction to sEH protein structure and function. sEH evolution and gene structure are then discussed before human small nucleotide polymorphisms and mammalian gene expression are described in the context of several disease models. The review ends with an overview of studies that have employed the sEH knockout mouse model. PMID:23701967

  18. Isolation and characterization of Xenopus soluble epoxide hydrolase.

    PubMed

    Purba, Endang R; Oguro, Ami; Imaoka, Susumu

    2014-07-01

    Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

  19. Soluble epoxide hydrolase inhibitory activity of anthraquinone components from Aloe.

    PubMed

    Sun, Ya Nan; Kim, Jang Hoon; Li, Wei; Jo, A Reum; Yan, Xi Tao; Yang, Seo Young; Kim, Young Ho

    2015-10-15

    Aloe is a short-stemmed succulent herb widely used in traditional medicine to treat various diseases and as raw material in cosmetics and heath foods. In this study, we isolated and identified two new anthraquinone derivatives, aloinoside C (6) and aloinoside D (7), together with six known compounds from an aqueous dissolved Aloe exudate. Their structures were identified by spectroscopic analysis. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were evaluated. Compounds 1-8 inhibited sEH activity potently, with IC50 values ranging from 4.1±0.6 to 41.1±4.2 μM. A kinetic analysis of compounds 1-8 revealed that the inhibitory actions of compounds 1, 6 and 8 were non-competitive, whereas those of compounds 2-5 and 7 were the mixed-type. Molecular docking increases our understanding of receptor-ligand binding of all compounds. These results demonstrate that compounds 1-8 from Aloe are potential sEH inhibitors.

  20. Co-purification of microsomal epoxide hydrolase with the warfarin-sensitive vitamin K1 oxide reductase of the vitamin K cycle.

    PubMed

    Guenthner, T M; Cai, D; Wallin, R

    1998-01-15

    Vitamin K1 oxide reductase activity has been partially purified from rat liver microsomes. A three-step procedure produced a preparation in which warfarin-sensitive vitamin K1 oxide reductase activity was 118-fold enriched over the activity in intact rat liver microsomes. A major component of the multi-protein mixture was identified as a 50 kDa protein that strongly cross-reacts with antiserum prepared against homogeneous rat liver microsomal epoxide hydrolase. The reductase preparation also had a high level or epoxide hydrolase activity against two xenobiotic epoxide substrates. The K(m) values for hydrolysis by the reductase preparation were similar to those for homogeneous microsomal epoxide hydrolase itself, and the specific hydrolase activities of the reductase preparation were 25-35% of the specific activities measured for the homogeneous hydrolase preparation. Antibodies prepared against homogeneous microsomal epoxide hydrolase inhibited up to 80% of reductase activity of the reductase preparation. Homogeneous microsomal epoxide hydrolase had no vitamin K1 oxide reductase activity. This evidence suggests that microsomal epoxide hydrolase, or a protein that is very similar to it, is a major functional component of a multi-protein complex that is responsible for vitamin K1 oxide reduction in rat liver microsomes.

  1. Soluble epoxide hydrolase: a novel therapeutic target in stroke

    PubMed Central

    Zhang, Wenri; Koerner, Ines P; Noppens, Ruediger; Grafe, Marjorie; Tsai, Hsing-Ju; Morisseau, Christophe; Luria, Ayala; Hammock, Bruce D; Falck, John R; Alkayed, Nabil J

    2009-01-01

    The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke. PMID:17440491

  2. Expression and characterization of an epoxide hydrolase from Anopheles gambiae with high activity on epoxy fatty acids

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Hammock, Bruce D.

    2014-01-01

    In insects, epoxide hydrolases (EHs) play critical roles in the metabolism of xenobiotic epoxides from the food resources and in the regulation of endogenous chemical mediators, such as juvenile hormones. Using the baculovirus expression system, we expressed and characterized an epoxide hydrolase from Anopheles gambiae (AgEH) that is distinct in evolutionary history from insect juvenile hormone epoxide hydrolases (JHEHs). We partially purified the enzyme by ion exchange chromatography and isoelectric focusing. The experimentally determined molecular weight and pI were estimated to be 35kD and 6.3 respectively, different than the theoretical ones. The AgEH had the greatest activity on long chain epoxy fatty acids such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and 9,10-epoxy-12Z-octadecenoic acids (9,10-EpOME or leukotoxin) among the substrates evaluated. Juvenile hormone III, a terpenoid insect growth regulator, was the next best substrate tested. The AgEH showed kinetics comparable to the mammalian soluble epoxide hydrolases, and the activity could be inhibited by AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based inhibitor designed to inhibit the mammalian soluble epoxide hydrolases. The rabbit serum generated against the soluble epoxide hydrolase of Mus musculus can both cross-react with natural and denatured forms of the AgEH, suggesting immunologically they are similar. The study suggests there are mammalian sEH homologs in insects, and epoxy fatty acids may be important chemical mediators in insects. PMID:25173592

  3. Identification and characterization of a new epoxide hydrolase from mouse liver microsomes.

    PubMed

    Guenthner, T M; Oesch, F

    1983-12-25

    A new microsomal epoxide hydrolase (mEH2) has been identified and characterized. This enzyme has properties which distinguish it from previously described cytosolic (cEH) or membrane-bound (mEH1) epoxide hydrolases. The enzyme is an integral microsomal protein which is not dissociated from the membrane by repeated washing, high ionic strength salt, or chaotropic agent solutions, or by sonication. It is very different from the normally described microsomal epoxide hydrolase (mEH1) as shown by its different substrate specificity and kinetic properties and by immunological criteria. In contrast to the hitherto described microsomal epoxide hydrolase, mEH1, the new enzyme effectively catalyzes the hydration of transdisubstituted oxiranes such as trans-stilbene oxide and trans-beta-ethyl styrene oxide and has no appreciable activity toward benzo(a)pyrene 4,5-oxide. It is also structurally distinct, in that it does not cross-react with antibodies raised against the normally described microsomal epoxide hydrolase mEH1. This newly described microsomal epoxide hydrolase probably represents an important factor in the control of reactive epoxides; its location in the membrane ensures access to lipophilic epoxides generated by membrane-bound monooxygenases, and its substrate specificity is such that it can hydrolyze epoxides poorly metabolized by the previously described microsomal epoxide hydrolase.

  4. Synthesis and structure-activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors

    SciTech Connect

    Pecic, Stevan; Pakhomova, Svetlana; Newcomer, Marcia E.; Morisseau, Christophe; Hammock, Bruce D.; Zhu, Zhengxiang; Rinderspacher, Alison; Deng, Shi-Xian

    2013-09-27

    A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure–activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.

  5. Structure of a soluble epoxide hydrolase identified in Trichoderma reesei.

    PubMed

    Wilson, Carolina; De Oliveira, Gabriel S; Adriani, Patrícia P; Chambergo, Felipe S; Dias, Marcio V B

    2017-08-01

    Epoxide hydrolases (EHs) are enzymes that have high biotechnological interest for the fine and transformation industry. Several of these enzymes have enantioselectivity, which allows their application in the separation of enantiomeric mixtures of epoxide substrates. Although two different families of EHs have been described, those that have the α/β-hidrolase fold are the most explored for biotechnological purpose. These enzymes are functionally very well studied, but only few members have three-dimensional structures characterised. Recently, a new EH from the filamentous fungi Trichoderma reseei (TrEH) has been discovered and functionally studied. This enzyme does not have high homology to any other EH structure and have an enatiopreference for (S)-(-) isomers. Herein we described the crystallographic structure of TrEH at 1.7Å resolution, which reveals features of its tertiary structure and active site. TrEH has a similar fold to the other soluble epoxide hydrolases and has the two characteristic hydrolase and cap domains. The enzyme is predominantly monomeric in solution and has also been crystallised as a monomer in the asymmetric unit. Although the catalytic residues are conserved, several other residues of the catalytic groove are not, and might be involved in the specificity for substrates and in the enantioselectivy of this enzyme. In addition, the determination of the crystallographic structure of TrEH might contribute to the rational site direct mutagenesis to generate an even more stable enzyme with higher efficiency to be used in biotechnological purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Discovery of enantioselectivity of urea inhibitors of soluble epoxide hydrolase.

    PubMed

    Manickam, Manoj; Pillaiyar, Thanigaimalai; Boggu, PullaReddy; Venkateswararao, Eeda; Jalani, Hitesh B; Kim, Nam-Doo; Lee, Seul Ki; Jeon, Jang Su; Kim, Sang Kyum; Jung, Sang-Hun

    2016-07-19

    Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) in the metabolic pathway of arachidonic acid and has been considered as an important therapeutic target for chronic diseases such as hypertension, diabetes and inflammation. Although many urea derivatives are known as sEH inhibitors, the enantioselectivity of the inhibitors is not highlighted in spite of the stereoselective hydrolysis of EETs by sEH. In an effort to explore the importance of enantioselectivity in the urea scaffold, a series of enantiomers with the stereocenter adjacent to the urea nitrogen atom were prepared. The selectivity of enantiomers of 1-(α-alkyl-α-phenylmethyl)-3-(3-phenylpropyl)ureas showed wide range differences up to 125 fold with the low IC50 value up to 13 nM. The S-configuration with planar phenyl and small alkyl groups at α-position is crucial for the activity and selectivity. However, restriction of the free rotation of two α-groups with indan-1-yl or 1,2,3,4-tetrahydronaphthalen-1-yl moiety abolishes the selectivity between the enantiomers, despite the increase in activity up to 13 nM. The hydrophilic group like sulfonamido group at para position of 3-phenylpropyl motif of 1-(α-alkyl-α-phenylmethyl-3-(3-phenylpropyl)urea improves the activity as well as enantiomeric selectivity. All these ureas are proved to be specific inhibitor of sEH without inhibition against mEH. Copyright © 2016. Published by Elsevier Masson SAS.

  7. Peripheral FAAH and soluble epoxide hydrolase inhibitors are synergistically antinociceptive.

    PubMed

    Sasso, Oscar; Wagner, Karen; Morisseau, Christophe; Inceoglu, Bora; Hammock, Bruce D; Piomelli, Daniele

    2015-07-01

    We need better medicines to control acute and chronic pain. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) catalyze the deactivating hydrolysis of two classes of bioactive lipid mediators--fatty acid ethanolamides (FAEs) and epoxidized fatty acids (EpFAs), respectively--which are biogenetically distinct but share the ability to attenuate pain responses and inflammation. In these experiments, we evaluated the antihyperalgesic activity of small-molecule inhibitors of FAAH and sEH, administered alone or in combination, in two pain models: carrageenan-induced hyperalgesia in mice and streptozocin-induced allodynia in rats. When administered separately, the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea (TPPU) and the peripherally restricted FAAH inhibitor URB937 were highly active in the two models. The combination TPPU plus URB937 was markedly synergistic, as assessed using isobolographic analyses. The results of these experiments reveal the existence of a possible functional crosstalk between FAEs and EpFAs in regulating pain responses. Additionally, the results suggest that combinations of sEH and FAAH inhibitors might be exploited therapeutically to achieve greater analgesic efficacy.

  8. The effects of modulation of microsomal epoxide hydrolase activity on microsome-catalyzed activation of benzo[alpha]pyrene and its covalent binding to DNA.

    PubMed

    Guenthner, T M; Oesch, F

    1981-01-01

    The effects of modulation of microsomal epoxide hydrolase activity on the binding of calf thymus DNA of benzo[alpha]pyrene metabolically activated by rat liver microsomes were investigated. In systems where microsomal epoxide hydrolase levels were not manipulated, 2 major bound species, one derived from 9-hydroxybenzo[alpha]pyrene and the other derived from benzo[alpha]pyrene 7,8-dihydrodiol, were found in approximately equivalent amounts. When epoxide hydrolase levels were increased, either by addition in vitro of purified enzyme or by induction in vivo by trans-stilbene oxide, the binding of the benzo[alpha]pyrene 7,8-dihydrodiol product was increased, while the binding of the 9-hydroxybenzo[alpha]pyrene product was practically eliminated. When microsomal epoxide hydrolase activity was decreased by selective inhibition with low concentrations of 1,1,1-trichloropropene 2,3-oxide, the binding of the species derived from 9-hydroxybenzo[alpha]pyrene was increased several-fold, while that of the species derived from benzo[alpha]pyrene 7,8-dihydrodiol was greatly decreased. The results indicate that the binding species derived from 9-hydroxybenzo[alpha]pyrene is formed through a metabolic pathway leading to an epoxide which is a substrate of microsomal epoxide hydrolase and that microsomal epoxide hydrolase is important in regulating the pattern of binding of individual microsomally-formed benzo[alpha]pyrene metabolites to DNA.

  9. Alteration of the mutagenicity 3,3'-dichlorobenzidine by modifiers of rat hepatic epoxide hydrolase activity

    SciTech Connect

    Iba, M.M.

    1986-03-05

    The involvement of arene oxides in the activation of benzidines was assessed by examining the effect of (I) the epoxide hydrolase inhibitor trichloropropylene oxide (TCPO), (II) purified rat liver microsomal (P) epoxide hydrolase (EH), and (III) pretreatment of rats with phenobarbital (PB) on hepatic Sg- or P-catalyzed mutagenicity of benzidine (BZ) and 3,3'-dichlorobenzidine (DCB) to Salmonella TA 98. When catalyzed by Sg from untreated rats, the mutagenicity of DCB and BZ was 601 +/- 101 and 79 +/- 25 (His/sup +/ revertants/plate) respectively, but was 345 +/- 55 and 226 +/- 30 respectively, when catalyzed by microsomes (P) from untreated rats. PB-pretreatment enhanced the Sg-catalyzed mutagenicity of DCB and BZ (2.3-fold and 1.7-fold, respectively) and the P-catalyzed mutagenicity of DCB (1.7-fold), but totally inhibited the P-catalyzed mutagenicity of BZ. In TCPO-supplemented activating systems from PB-pretreated rats, the mutagenicity of DCB was enhanced in both Sg and P (1.9-fold and 1.6-fold, respectively), whereas that of BZ was unchanged. Added EH enhanced the P-catalyzed mutagenicity of DCB (1.4-fold) but had no effect on that of BZ, suggesting that the activity of the enzyme on DCB metabolites may not be entirely detoxifying. The data suggest that epoxidation may contribute to the activation of DCB but not BZ.

  10. Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200.

    PubMed

    Kotik, Michael; Kyslík, Pavel

    2006-02-01

    Purification of a novel enantioselective epoxide hydrolase from Aspergillus niger M200 has been achieved using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, and size-exclusion chromatography, in conjunction with two additional chromatographic steps employing hydroxylapatite, and Mimetic Green. The enzyme was purified 186-fold with a yield of 15%. The apparent molecular mass of the enzyme was determined to be 77 kDa under native conditions and 40 kDa under denaturing conditions, implying a dimeric structure of the native enzyme. The isoelectric point of the enzyme was estimated to be 4.0 by isoelectric focusing electrophoresis. The enzyme has a broad substrate specificity with highest specificities towards tert-butyl glycidyl ether, para-nitrostyrene oxide, benzyl glycidyl ether, and styrene oxide. Enantiomeric ratios of 30 to more than 100 were determined for the hydrolysis reactions of 4 epoxidic substrates using the purified enzyme at a reaction temperature of 10 degrees C. Product inhibition studies suggest that the enzyme is able to differentiate to a high degree between the (R)-diol and (S)-diol product of the hydrolysis reaction with tert-butyl glycidyl ether as the substrate. The highest activity of the enzyme was at 42 degrees C and a pH of 6.8. Six peptide sequences, which were obtained by cleavage of the purified enzyme with trypsin and mass spectrometry analysis of the tryptic peptides, show high similarity with corresponding sequences originated from the epoxide hydrolase from Aspergillus niger LCP 521.

  11. Enantioselective hydrolysis of racemic epichlorohydrin using an epoxide hydrolase from Novosphingobium aromaticivorans.

    PubMed

    Woo, Jung-Hee; Hwang, Young-Ok; Kang, Ji-Hyun; Lee, Hyun Sook; Kim, Sang-Jin; Kang, Sung Gyun

    2010-09-01

    Previously we reported that an epoxide hydrolase (EHase) from Novosphingobium aromaticivorans could preferentially hydrolyze (R)-styrene oxide. In this study, we demonstrate that the purified NEH could be also effective in chiral resolution of racemic epichlorohydrin (ECH). Particularly, the purified NEH showed excellent hydrolyzing activity toward ECH to complete the reaction at a short period of incubation time. Enantiopure (S)-ECH could be obtained with a high enantiopurity of more than 99.99% enantiomeric excess (ee) and yield of 20.7% (theoretical, 50%). The chiral resolution of the purified NEH toward ECH was not susceptible to substrate inhibition by 500 mM racemic ECH.

  12. Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.

    PubMed

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-08-01

    Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool.

  13. Inhibition of Chronic Pancreatitis and Murine Pancreatic Intraepithelial Neoplasia by a Dual Inhibitor of c-RAF and Soluble Epoxide Hydrolase in LSL-KrasG12D/Pdx-1-Cre Mice

    PubMed Central

    LIAO, JIE; HWANG, SUNG HEE; LI, HAONAN; LIU, JUN-YAN; HAMMOCK, BRUCE D.; YANG, GUANG-YU

    2016-01-01

    Mutation of Kirsten rat sarcoma viral oncogene homolog (KRAS) and chronic pancreatitis are the most common pathogenic events involved in human pancreatic carcinogenesis. In the process of long-standing chronic inflammation, aberrant metabolites of arachidonic acid play a crucial role in promoting carcinogenesis, in which the soluble epoxide hydrolase (sEH), as a pro-inflammatory enzyme, generally inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs). Herein, we determined the effect of our newly-synthesized novel compound trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM), a dual inhibitor of sEH and RAF1 proto-oncogene serine/threonine kinase (c-RAF), on inhibiting the development of pancreatitis and pancreatic intraepithelial neoplasia (mPanIN) in LSL-KrasG12D/Pdx1-Cre mice. The results showed that t-CUPM significantly reduced the severity of chronic pancreatitis, as measured by the extent of acini loss, inflammatory cell infiltration and stromal fibrosis. The progression of low-grade mPanIN I to high-grade mPanIN II/III was significantly suppressed. Inhibition of mutant Kras-transmitted phosphorylation of mitogen-activated protein kinase’s kinase/extracellular signal-regulated kinases was demonstrated in pancreatic tissues by western blots. Quantitative real-time polymerase chain reaction analysis revealed that t-CUPM treatment significantly reduced the levels of inflammatory cytokines including tumor necrosis facor-α, monocyte chemoattractant protein-1, as well as vascular adhesion molecule-1, and the levels of Sonic hedgehog and Gli transcription factor (Hedgehog pathway). Analysis of the eicosanoid profile revealed a significant increase of the EETs/dihydroxyeicosatrienoic acids ratio, which further confirmed sEH inhibition by t-CUPM. These results indicate that simultaneous inhibition of sEH and c-RAF by t-CUPM is important in preventing chronic pancreatitis and carcinogenesis

  14. Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

    PubMed

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-09-01

    The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

  15. Ingestion of the epoxide hydrolase inhibitor AUDA modulates immune responses of the mosquito, Culex quinquefasciatus during blood feeding.

    PubMed

    Xu, Jiawen; Morisseau, Christophe; Yang, Jun; Lee, Kin Sing Stephen; Kamita, Shizuo G; Hammock, Bruce D

    2016-09-01

    Epoxide hydrolases (EHs) are enzymes that play roles in metabolizing xenobiotic epoxides from the environment, and in regulating lipid signaling molecules, such as juvenile hormones in insects and epoxy fatty acids in mammals. In this study we fed mosquitoes with an epoxide hydrolase inhibitor AUDA during artificial blood feeding, and we found the inhibitor increased the concentration of epoxy fatty acids in the midgut of female mosquitoes. We also observed ingestion of AUDA triggered early expression of defensin A, cecropin A and cecropin B2 at 6 h after blood feeding. The expression of cecropin B1 and gambicin were not changed more than two fold compared to controls. The changes in gene expression were transient possibly because more than 99% of the inhibitor was metabolized or excreted at 42 h after being ingested. The ingestion of AUDA also affected the growth of bacteria colonizing in the midgut, but did not affect mosquito longevity, fecundity and fertility in our laboratory conditions. When spiked into the blood, EpOMEs and DiHOMEs were as effective as the inhibitor AUDA in reducing the bacterial load in the midgut, while EETs rescued the effects of AUDA. Our data suggest that epoxy fatty acids from host blood are immune response regulators metabolized by epoxide hydrolases in the midgut of female mosquitoes, inhibition of which causes transient changes in immune responses, and affects growth of microbes in the midgut.

  16. The role of human glutathione transferases and epoxide hydrolases in the metabolism of xenobiotics.

    PubMed Central

    Seidegård, J; Ekström, G

    1997-01-01

    Human glutathione transferases (GSTs) are a multigene family of enzymes that are involved in the metabolism of a wide range of electrophilic compounds of both exogenous and endogenous origin. GSTs are generally recognized as detoxifying enzymes by catalyzing the conjugation of these compounds with glutathione, but they may also be involved in activation of some carcinogens. The memmalian GSTs can be differentiated into four classes of cytosolic enzymes and two membrane bound enzymes. Human epoxide hydrolases (EHs) catalyze the addition of water to epoxides to form the corresponding dihydrodiol. The enzymatic hydration is essentially irreversible and produces mainly metabolites of lower reactivity that can be conjugated and excreted. The reaction of EHs is therefore generally regarded as detoxifying. The mammalian EHs can be distinguished by their physical and enzymatic properties. Microsomal EH (mEH) exhibits a broad substrate specificity, while the soluble EH (sEH) is an enzyme with a "complementary" substrate specificity to mEH. Cholesterol EH and leukotriene A4 hydrolase are two EHs with very limited substrate specificity. The activities of either GSTs or EHs expressed in vivo exhibit a relatively large interindividual variation, which might be explained by induction, inhibition, or genetic factors. These variations in levels or activities of individual isoenzymes are of importance with respect to an individual's susceptibility to genotoxic effects. This article gives a general overview of GSTs and EHs, discussing the modulation of activities, determination of these enzymes ex vivo, and the polymorphic expression of some isoenzymes. PMID:9255563

  17. Disrupting Dimerization Translocates Soluble Epoxide Hydrolase to Peroxisomes.

    PubMed

    Nelson, Jonathan W; Das, Anjali J; Barnes, Anthony P; Alkayed, Nabil J

    2016-01-01

    The epoxyeicosatrienoic acid (EET) neutralizing enzyme soluble epoxide hydrolase (sEH) is a neuronal enzyme, which has been localized in both the cytosol and peroxisomes. The molecular basis for its dual localization remains unclear as sEH contains a functional peroxisomal targeting sequence (PTS). Recently, a missense polymorphism was identified in human sEH (R287Q) that enhances its peroxisomal localization. This same polymorphism has also been shown to generate weaker sEH homo-dimers. Taken together, these observations suggest that dimerization may mask the sEH PTS and prevent peroxisome translocation. In the current study, we test the hypothesis that dimerization is a key regulator of sEH subcellular localization. Specifically, we altered the dimerization state of sEH by introducing substitutions in amino acids responsible for the dimer-stabilizing salt-bridge. Green Fluorescent Protein (GFP) fusions of each of mutants were co-transfected into mouse primary cultured cortical neurons together with a PTS-linked red fluorescent protein to constitutively label peroxisomes. Labeled neurons were analyzed using confocal microscopy and co-localization of sEH with peroxisomes was quantified using Pearson's correlation coefficient. We find that dimer-competent sEH constructs preferentially localize to the cytosol, whereas constructs with weakened or disrupted dimerization were preferentially targeted to peroxisomes. We conclude that the sEH dimerization status is a key regulator of its peroxisomal localization.

  18. Soluble Epoxide Hydrolase Inhibitory Activity of Selaginellin Derivatives from Selaginella tamariscina.

    PubMed

    Kim, Jang Hoon; Cho, Chong Woon; Tai, Bui Huu; Yang, Seo Young; Choi, Gug-Seoun; Kang, Jong Seong; Kim, Young Ho

    2015-12-02

    Selaginellin derivatives 1-3 isolated from Selaginella tamariscina were evaluated for their inhibition of soluble epoxide hydrolase (sEH) to demonstrate their potential for the treatment of cardiovascular disease. All selaginellin derivatives (1-3) inhibited sEH enzymatic activity and PHOME hydrolysis, in a dose-dependent manner, with IC50 values of 3.1 ± 0.1, 8.2 ± 2.2, and 4.2 ± 0.2 μM, respectively. We further determined that the derivatives function as non-competitive inhibitors. Moreover, the predicted that binding sites and interaction between 1-3 and sEH were solved by docking simulations. According to quantitative analysis, 1-3 were confirmed to have high content in the roots of S. tamariscina; among them, selaginellin 3 exhibited the highest content of 189.3 ± 0.0 μg/g.

  19. Exported Epoxide Hydrolases Modulate Erythrocyte Vasoactive Lipids during Plasmodium falciparum Infection

    PubMed Central

    Dalmia, Varun K.

    2016-01-01

    ABSTRACT Erythrocytes are reservoirs of important epoxide-containing lipid signaling molecules, including epoxyeicosatrienoic acids (EETs). EETs function as vasodilators and anti-inflammatory modulators in the bloodstream. Bioactive EETs are hydrolyzed to less active diols (dihydroxyeicosatrienoic acids) by epoxide hydrolases (EHs). The malaria parasite Plasmodium falciparum infects host red blood cells (RBCs) and exports hundreds of proteins into the RBC compartment. In this study, we show that two parasite epoxide hydrolases, P. falciparum epoxide hydrolases 1 (PfEH1) and 2 (PfEH2), both with noncanonical serine nucleophiles, are exported to the periphery of infected RBCs. PfEH1 and PfEH2 were successfully expressed in Escherichia coli, and they hydrolyzed physiologically relevant erythrocyte EETs. Mutations in active site residues of PfEH1 ablated the ability of the enzyme to hydrolyze an epoxide substrate. Overexpression of PfEH1 or PfEH2 in parasite-infected RBCs resulted in a significant alteration in the epoxide fatty acids stored in RBC phospholipids. We hypothesize that the parasite disruption of epoxide-containing signaling lipids leads to perturbed vascular function, creating favorable conditions for binding and sequestration of infected RBCs to the microvascular endothelium. PMID:27795395

  20. Soluble epoxide hydrolase limits mechanical hyperalgesia during inflammation

    PubMed Central

    2011-01-01

    Background Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation. Results In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH-/- mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH-/- mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice. Conclusion Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects. PMID:21970373

  1. Naphthalene cytotoxicity in microsomal epoxide hydrolase deficient mice

    PubMed Central

    Carratt, SA; Morin, D; Buckpitt, AR; Edwards, PC; Van Winkle, LS

    2016-01-01

    Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (ie 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4 hours). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure. PMID:26840748

  2. Naphthalene cytotoxicity in microsomal epoxide hydrolase deficient mice.

    PubMed

    Carratt, S A; Morin, D; Buckpitt, A R; Edwards, P C; Van Winkle, L S

    2016-03-30

    Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Cytochrome P450 epoxygenases, soluble epoxide hydrolase, and the regulation of cardiovascular inflammation.

    PubMed

    Deng, Yangmei; Theken, Katherine N; Lee, Craig R

    2010-02-01

    The cytochrome P450 (CYP) epoxygenase enzymes CYP2J and CYP2C catalyze the epoxidation of arachidonic acid to epoxyeicosatrienoic acids (EETs), which are rapidly hydrolyzed to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). It is well-established that CYP epoxygenase-derived EETs possess potent vasodilatory effects; however, the cellular effects of EETs and their regulation of various inflammatory processes have become increasingly appreciated in recent years, suggesting that the role of this pathway in the cardiovascular system extends beyond the maintenance of vascular tone. In particular, CYP epoxygenase-derived EETs inhibit endothelial activation and leukocyte adhesion via attenuation of nuclear factor-kappaB activation, inhibit hemostasis, protect against myocardial ischemia-reperfusion injury, and promote endothelial cell survival via modulation of multiple cell signaling pathways. Thus, the CYP epoxygenase pathway is an emerging target for pharmacological manipulation to enhance the cardiovascular protective effects of EETs. This review will focus on the role of the CYP epoxygenase pathway in the regulation of cardiovascular inflammation and (1) describe the functional impact of CYP epoxygenase-derived EET biosynthesis and sEH-mediated EET hydrolysis on key inflammatory process in the cardiovascular system, (2) discuss the potential relevance of this pathway to pathogenesis and treatment of cardiovascular disease, and (3) identify areas for future research.

  4. Contribution of hydrolase and phosphatase domains in soluble epoxide hydrolase to vascular endothelial growth factor expression and cell growth.

    PubMed

    Oguro, Ami; Sakamoto, Koichi; Suzuki, Sachiko; Imaoka, Susumu

    2009-12-01

    Soluble epoxide hydrolase (sEH) is an important pharmacological target because it metabolizes potent bioactive substrates, epoxyeicosatrienoinc acids (EETs) and other lipid epoxide. EETs have a variety of biological functions including angiogenesis and cancer metastasis. However, the regulation and physiological function of sEH is not well understood. In this study, we found that hypoxia significantly suppressed the expression of sEH in mouse liver and a human hepatoma cell line, Hep3B. Hypoxia promotes the proliferation of vascular endothelial cells or carcinoma cells. Knockdown of sEH in Hep3B cells induced vascular endothelial growth factor (VEGF) mRNA and cell growth, both of which were suppressed by overexpression of sEH. sEH has phosphatase activity as well as epoxide hydrolase (EH) activity. We prepared mutant clones which lacking EH or phosphatase activity using the amino acid change Asp335Ser or Asp9Ala, respectively. The effects of WT sEH on cell growth were lost by mutation of either the EH domain or phosphatase domain. However, mutation of the phosphatase domain but not EH domain did not influence the expression of VEGF. These results suggest that sEH plays an important role in the physiology of cells including proliferation and that the epoxide hydrolase and phosphatase domains of sEH have different biological functions.

  5. Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation

    NASA Astrophysics Data System (ADS)

    Davis, Benjamin B.; Thompson, David A.; Howard, Laura L.; Morisseau, Christophe; Hammock, Bruce D.; Weiss, Robert H.

    2002-02-01

    Atherosclerosis, in its myriad incarnations the foremost killer disease in the industrialized world, is characterized by aberrant proliferation of vascular smooth muscle (VSM) cells in part as a result of the recruitment of inflammatory cells to the blood vessel wall. The epoxyeicosatrienoic acids are synthesized from arachidonic acid in a reaction catalyzed by the cytochrome P450 system and are vasoactive substances. Metabolism of these compounds by epoxide hydrolases results in the formation of compounds that affect the vasculature in a pleiotropic manner. As an outgrowth of our observations that urea inhibitors of the soluble epoxide hydrolase (sEH) reduce blood pressure in spontaneously hypertensive rats as well as the findings of other investigators that these compounds possess antiinflammatory actions, we have examined the effect of sEH inhibitors on VSM cell proliferation. We now show that the sEH inhibitor 1-cyclohexyl-3-dodecyl urea (CDU) inhibits human VSM cell proliferation in a dose-dependent manner and is associated with a decrease in the level of cyclin D1. In addition, cis-epoxyeicosatrienoic acid mimics the growth-suppressive activity of CDU; there is no evidence of cellular toxicity or apoptosis in CDU-treated cells when incubated with 20 μM CDU for up to 48 h. These results, in light of the antiinflammatory and antihypertensive properties of these compounds that have been demonstrated already, suggest that the urea class of sEH inhibitors may be useful for therapy for diseases such as hypertension and atherosclerosis characterized by exuberant VSM cell proliferation and vascular inflammation.

  6. N-terminal domain of soluble epoxide hydrolase negatively regulates the VEGF-mediated activation of endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Hammock, Bruce D.; Su, Kou-Hui; Morisseau, Christophe; Kou, Yu Ru; Imaoka, Susumu; Oguro, Ami; Shyue, Song-Kun; Zhao, Jin-Feng; Lee, Tzong-Shyuan

    2012-01-01

    Aims The mammalian soluble epoxide hydrolase (sEH) has both an epoxide hydrolase and a phosphatase domain. The role of sEH hydrolase activity in the metabolism of epoxyeicosatrienoic acids (EETs) and the activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) has been well defined. However, far less is known about the role of sEH phosphatase activity in eNOS activation. In the present study, we investigated whether the phosphatase domain of sEH was involved in the eNOS activation in ECs. Methods and results The level of eNOS phosphorylation in aortas is higher in the sEH knockout (sEH−/−) mice than in wild-type mice. In ECs, pharmacological inhibition of sEH phosphatase or overexpressing sEH with an inactive phosphatase domain enhanced vascular endothelial growth factor (VEGF)-induced NO production and eNOS phosphorylation. In contrast, overexpressing the phosphatase domain of sEH prevented the VEGF-mediated NO production and eNOS phosphorylation at Ser617, Ser635, and Ser1179. Additionally, treatment with VEGF induced a c-Src kinase-dependent increase in transient tyrosine phosphorylation of sEH and the formation of a sEH–eNOS complex, which was abolished by treatment with a c-Src kinase inhibitor, PP1, or the c-Src dominant-negative mutant K298M. We also demonstrated that the phosphatase domain of sEH played a key role in VEGF-induced angiogenesis by detecting the tube formation in ECs and neovascularization in Matrigel plugs in mice. Conclusion In addition to epoxide hydrolase activity, phosphatase activity of sEH plays a pivotal role in the regulation of eNOS activity and NO-mediated EC functions. PMID:22072631

  7. Optimization of Amide-Based Inhibitors of Soluble Epoxide Hydrolase with Improved Water Solubility

    PubMed Central

    Kim, In-Hae; Heirtzler, Fenton R.; Morisseau, Christophe; Nishi, Kosuke; Tsai, Hsing-Ju; Hammock, Bruce D.

    2006-01-01

    Soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in the regulation of blood pressure and inflammation. 1,3-Disubstituted ureas with a polar group located on the fifth atom from the carbonyl group of urea function are active inhibitors of sEH both in vitro and in vivo. However, their limited solubility in water and relatively high melting point lead to difficulties in formulating the compounds and poor in vivo efficacy. To improve these physical properties, the effect of structural modification of the urea pharmacophore on the inhibition potencies, water solubilities, octanol/water partition coefficients (log P), and melting points of a series of compounds was evaluated. For murine sEH, no loss of inhibition potency was observed when the urea pharmacophore was modified to an amide function, while for human sEH 2.5-fold decreased inhibition was obtained in the amide compounds. In addition, a NH group on the right side of carbonyl group of the amide pharmacophore substituted with an adamantyl group (such as compound 14) and a methylene carbon present between the adamantyl and amide groups were essential to produce potent inhibition of sEH. The resulting amide inhibitors have 10–30-fold better solubility and lower melting point than the corresponding urea compounds. These findings will facilitate synthesis of sEH inhibitors that are easier to formulate and more bioavailable. PMID:15887969

  8. Detoxication strategy of epoxide hydrolase-the basis for a novel threshold for definable genotoxic carcinogens.

    PubMed

    Oesch, Franz; Hengstler, Jan Georg; Arand, Michael

    2004-01-01

    From our recent work on the three-dimensional structure of epoxide hydrolases we theoretically deduced the likelihood of a two-step catalytic mechanism that we and others have subsequently experimentally confirmed. Analysis of the rate of the two steps by us and by others show that the first step-responsible for removal of the reactive epoxide from the system-works extraordinarily fast (typically three orders of magnitude faster than the second step), sucking up the epoxide like a sponge. Regeneration of the free enzyme (the second step of the catalytic mechanism) is slow. This becomes a toxicological problem only at doses of the epoxide that titrate the enzyme out. Our genotoxicity work shows that indeed this generates a practical threshold below which no genotoxicity is observed. This shows that-contrary to old dogma-practical thresholds exist for definable genotoxic carcinogens.

  9. Trigger factor assisted folding of the recombinant epoxide hydrolases identified from C. pelagibacter and S. nassauensis.

    PubMed

    Saini, Priya; Wani, Shadil Ibrahim; Kumar, Ranjai; Chhabra, Ravneet; Chimni, Swapandeep Singh; Sareen, Dipti

    2014-12-01

    Epoxide hydrolases (EHs), are enantioselective enzymes as they catalyze the kinetic resolution of racemic epoxides into the corresponding enantiopure vicinal diols, which are useful precursors in the synthesis of chiral pharmaceutical compounds. Here, we have identified and cloned two putative epoxide hydrolase genes (cpeh and sneh) from marine bacteria, Candidatus pelagibacter ubique and terrestrial bacteria, Stackebrandtia nassauensis, respectively and overexpressed them in pET28a vector in Escherichia coli BL21(DE3). The CPEH protein (42kDa) was found to be overexpressed as inactive inclusion bodies while SNEH protein (40kDa) was found to form soluble aggregates. In this study, the recombinant CPEH was successfully transformed from insoluble aggregates to the soluble and functionally active form, using pCold TF vector, though with low EH activity. To prevent the soluble aggregate formation of SNEH, it was co-expressed with GroEL/ES chaperone and was also fused with trigger factor (TF) chaperone at its N-terminus. The TF chaperone-assisted correct folding of SNEH led to a purified active EH with a specific activity of 3.85μmol/min/mg. The pure enzyme was further used to biocatalyze the hydrolysis of 10mM benzyl glycidyl ether (BGE) and α-methyl styrene oxide (MSO) with an enantiomeric excess of the product (eep) of 86% and 73% in 30 and 15min, respectively. In conclusion, this is the first report about the heterologous expression of epoxide hydrolases using TF as a molecular chaperone in pCold TF expression vector, resulting in remarkable increase in the solubility and activity of the otherwise improperly folded recombinant epoxide hydrolases.

  10. A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol.

    PubMed

    Wu, Shijin; Shen, Jiajia; Zhou, Xiaoyun; Chen, Jianmeng

    2007-10-01

    Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35 degrees C, pH 7.5 with glucose and NH(4)Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.

  11. The soluble epoxide hydrolase determines cholesterol homeostasis by regulating AMPK and SREBP activity.

    PubMed

    Mangels, Nicole; Awwad, Khader; Wettenmann, Annika; Dos Santos, Laila Romagueira Bichara; Frömel, Timo; Fleming, Ingrid

    2016-09-01

    Inhibition or deletion of the soluble epoxide hydrolase (sEH) has been linked to reduced cholesterol and protection against atherosclerosis. This study set out to identify sEH substrate(s) or product(s), altered in livers from sEH(-/-) mice that contribute to these beneficial effects. In livers and isolated hepatocytes, deletion of sEH decreased expression of HMG CoA reductase, fatty acid synthase and low density lipoprotein receptor. Sterol regulatory element binding proteins (SREBPs) regulate the expression of all three enzymes and SREBP activation was attenuated in the absence of sEH. The effect was attributed to the AMPK-activated protein kinase (AMPK) which was activated in the absence of sEH. Livers from wild-type versus sEH(-/-) littermates contained significantly higher levels of the sEH substrate 12,13-epoxyoctadecenoic acid, which elicited AMPK activation, while the corresponding sEH product was inactive. Thus, AMPK activation and subsequent inhibition of SREBP can account for the altered expression of lipid metabolizing enzymes in sEH(-/-) mice.

  12. In vitro metabolism of the mammalian soluble epoxide hydrolase inhibitor, 1-cyclohexyl-3-dodecyl-urea.

    PubMed

    Watanabe, Takaho; Morisseau, Christophe; Newman, John W; Hammock, Bruce D

    2003-07-01

    The metabolism of the soluble epoxide hydrolase (sEH) inhibitor, 1-cyclohexyl-3-dodecyl-urea (CDU), was studied in rat and human hepatic microsomes. The microsomal metabolism of CDU enhanced sEH inhibition potency of the reaction mixture and resulted in the formation of several metabolites. During the course of this study, a sensitive and specific high-performance liquid chromatography with tandem mass spectrometry analytical method was developed to investigate simultaneously the production of these metabolites. In both rat and human hepatic microsomes, CDU was ultimately transformed into the corresponding omega-carboxylate; however, the rodent tissue appeared to perform this transformation more rapidly. After a 60-min incubation in rat hepatic microsomes, the percentage of residual CDU, the omega-carboxylate, and the intermediary omega-hydroxyl were about 20%, 20%, and 50%, respectively. Carbon monoxide inhibited the metabolism of CDU by rat hepatic microsomes, suggesting that the initial step is catalyzed by cytochrome P450. Further metabolism was enhanced by the addition of NAD, suggesting that dehydrogenases are associated with intermediate metabolic steps. Regardless, the ultimate product of microsomal metabolism, 12-(3-cyclohexyl-ureido)-dodecanoic acid, is also an excellent sEH inhibitor with several hundred-fold higher solubility, supporting the hypothesis that CDU has prodrug characteristics. These findings will facilitate the rational design and optimization of sEH inhibitors with better physical properties and improved metabolic stability.

  13. Soluble epoxide hydrolase in podocytes is a significant contributor to renal function under hyperglycemia.

    PubMed

    Bettaieb, Ahmed; Koike, Shinichiro; Hsu, Ming-Fo; Ito, Yoshihiro; Chahed, Samah; Bachaalany, Santana; Gruzdev, Artiom; Calvo-Rubio, Miguel; Lee, Kin Sing Stephen; Inceoglu, Bora; Imig, John D; Villalba, Jose M; Zeldin, Darryl C; Hammock, Bruce D; Haj, Fawaz G

    2017-11-01

    Diabetic nephropathy (DN) is the leading cause of renal failure, and podocyte dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH, encoded by Ephx2) is a conserved cytosolic enzyme whose inhibition has beneficial effects on renal function. The aim of this study is to investigate the contribution of sEH in podocytes to hyperglycemia-induced renal injury. Mice with podocyte-specific sEH disruption (pod-sEHKO) were generated, and alterations in kidney function were determined under normoglycemia, and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia. sEH protein expression increased in murine kidneys under HFD- and STZ-induced hyperglycemia. sEH deficiency in podocytes preserved renal function and glucose control and mitigated hyperglycemia-induced renal injury. Also, podocyte sEH deficiency was associated with attenuated hyperglycemia-induced renal endoplasmic reticulum (ER) stress, inflammation and fibrosis, and enhanced autophagy. Moreover, these effects were recapitulated in immortalized murine podocytes treated with a selective sEH pharmacological inhibitor. Furthermore, pharmacological-induced elevation of ER stress or attenuation of autophagy in immortalized podocytes mitigated the protective effects of sEH inhibition. These findings establish sEH in podocytes as a significant contributor to renal function under hyperglycemia. These data suggest that sEH is a potential therapeutic target for podocytopathies. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Alteration in plasma testosterone levels in male mice lacking soluble epoxide hydrolase.

    PubMed

    Luria, Ayala; Morisseau, Christophe; Tsai, Hsing-Ju; Yang, Jun; Inceoglu, Bora; De Taeye, Bart; Watkins, Steven M; Wiest, Michelle M; German, J Bruce; Hammock, Bruce D

    2009-08-01

    Soluble epoxide hydrolase (Ephx2, sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH(2)-terminal phosphatase activities. sEH converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), and the phosphatase activity is suggested to be involved in cholesterol metabolism. EETs participate in a wide range of biological functions, including regulation of vascular tone, renal tubular transport, cardiac contractility, and inflammation. Inhibition of sEH is a potential approach for enhancing the biological activity of EETs. Therefore, disruption of sEH activity is becoming an attractive therapeutic target for both cardiovascular and inflammatory diseases. To define the physiological role of sEH, we characterized a knockout mouse colony lacking expression of the Ephx2 gene. Lack of sEH enzyme is characterized by elevation of EET to DHET ratios in both the linoleate and arachidonate series in plasma and tissues of both female and male mice. In male mice, this lack of expression was also associated with decreased plasma testosterone levels, sperm count, and testicular size. However, this genotype was still able to sire litters. Plasma cholesterol levels also declined in this genotype. Behavior tests such as anxiety-like behavior and hedonic response were also examined in Ephx2-null and WT mice, as all can be related to hormonal changes. Null mice showed a level of anxiety with a decreased hedonic response. In conclusion, this study provides a broad biochemical, physiological, and behavioral characterization of the Ephx2-null mouse colony and suggests a mechanism by which sEH and its substrates may regulate circulating levels of testosterone through cholesterol biosynthesis and metabolism.

  15. Colorimetric assays for quantitative analysis and screening of epoxide hydrolase activity.

    PubMed

    Cedrone, F; Bhatnagar, T; Baratti, Jacques C

    2005-12-01

    Focusing on directed evolution to tailor enzymes as usable biocatalysts for fine chemistry, we have studied in detail several colorimetric assays for quantitative analysis of epoxide hydrolase (EH) activity. In particular, two assays have been optimized to characterize variants issued from the directed evolution of the EH from Aspergillus niger. Assays described in this paper are sufficiently reliable for quantitative screening of EH activity in microtiter plates and are low cost alternatives to GC or MS analysis. Moreover, they are usable for various epoxides and not restricted to a type of substrate, such as those amenable to assay by UV absorbancy. They can be used to assay EH activity on any epoxide and to directly assay enantioselectivity when both (R) and (S) substrates are available. The advantages and drawbacks of these two methods to assay EH activity of a large number of natural samples are summarized.

  16. Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

    PubMed Central

    Kong, Xu-Dong; Yuan, Shuguang; Li, Lin; Chen, She; Xu, Jian-He; Zhou, Jiahai

    2014-01-01

    Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates. PMID:25331869

  17. Signature motifs identify an Acinetobacter Cif virulence factor with epoxide hydrolase activity.

    PubMed

    Bahl, Christopher D; Hvorecny, Kelli L; Bridges, Andrew A; Ballok, Alicia E; Bomberger, Jennifer M; Cady, Kyle C; O'Toole, George A; Madden, Dean R

    2014-03-14

    Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

  18. Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity*

    PubMed Central

    Bahl, Christopher D.; Hvorecny, Kelli L.; Bridges, Andrew A.; Ballok, Alicia E.; Bomberger, Jennifer M.; Cady, Kyle C.; O'Toole, George A.; Madden, Dean R.

    2014-01-01

    Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii (“aCif”). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog (“aCifR”) and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections. PMID:24474692

  19. Structure-Based Optimization of Arylamides as Inhibitors of Soluble Epoxide Hydrolase

    SciTech Connect

    Eldrup, Anne B.; Soleymanzadeh, Fariba; Taylor, Steven J.; Muegge, Ingo; Farrow, Neil A.; Joseph, David; McKellop, Keith; Man, Chuk C.; Kukulka, Alison; De Lombaert, Stephane

    2009-11-04

    Inhibition of soluble epoxide hydrolase (sEH) is hypothesized to lead to an increase in circulating levels of epoxyeicosatrienoic acids, resulting in the potentiation of their in vivo pharmacological properties. As part of an effort to identify inhibitors of sEH with high and sustained plasma exposure, we recently performed a high throughput screen of our compound collection. The screen identified N-(3,3-diphenyl-propyl)-nicotinamide as a potent inhibitor of sEH. Further profiling of this lead revealed short metabolic half-lives in microsomes and rapid clearance in the rat. Consistent with these observations, the determination of the in vitro metabolic profile of N-(3,3-diphenyl-propyl)-nicotinamide in rat liver microsomes revealed extensive oxidative metabolism and a propensity for metabolite switching. Lead optimization, guided by the analysis of the solid-state costructure of N-(3,3-diphenyl-propyl)-nicotinamide bound to human sEH, led to the identification of a class of potent and selective inhibitors. An inhibitor from this class displayed an attractive in vitro metabolic profile and high and sustained plasma exposure in the rat after oral administration.

  20. Optimized Inhibitors of Soluble Epoxide Hydrolase Improve in Vitro Target Residence Time and in Vivo Efficacy

    PubMed Central

    2015-01-01

    Diabetes is affecting the life of millions of people. A large proportion of diabetic patients suffer from severe complications such as neuropathic pain, and current treatments for these complications have deleterious side effects. Thus, alternate therapeutic strategies are needed. Recently, the elevation of epoxy-fatty acids through inhibition of soluble epoxide hydrolase (sEH) was shown to reduce diabetic neuropathic pain in rodents. In this report, we describe a series of newly synthesized sEH inhibitors with at least 5-fold higher potency and doubled residence time inside both the human and rodent sEH enzyme than previously reported inhibitors. These inhibitors also have better physical properties and optimized pharmacokinetic profiles. The optimized inhibitor selected from this new series displayed improved efficacy of almost 10-fold in relieving pain perception in diabetic neuropathic rats as compared to the approved drug, gabapentin, and previously published sEH inhibitors. Therefore, these new sEH inhibitors could be an attractive alternative to treat diabetic neuropathy in humans. PMID:25079952

  1. Lipid sulfates and sulfonates are allosteric competitive inhibitors of the N-terminal phosphatase activity of the mammalian soluble epoxide hydrolase.

    PubMed

    Tran, Katherine L; Aronov, Pavel A; Tanaka, Hiromasa; Newman, John W; Hammock, Bruce D; Morisseau, Christophe

    2005-09-13

    The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with K(I) in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH.

  2. Erectogenic and Aphrodisiac Property of Moringa oleifera: Involvement of Soluble Epoxide Hydrolase Enzyme.

    PubMed

    Goswami, Sumanta Kumar; Inamdar, Mohammed Naseeruddin; Dethe, Shekhar M; Gururaj, Giligar M; Jamwal, Rohitash; Bhaskar, Anirban; Mundkinajeddu, Deepak; Agarwal, Amit

    2016-07-01

    Soluble epoxide hydrolase (sEH) inhibitors have been reported to improve penile erection; therefore, sEH could be useful for management of erectile dysfunction. Methanolic and aqueous extracts of 30 Indian medicinal plants were screened for their sEH inhibition potential. Fifteen extracts showed >50% inhibition when screened at 50 µg/mL in sEH inhibition assay. Methanolic extract of Moringa oleifera Lam. (Moringaceae) seeds (MEMO) was most potent with IC50 1.7 ± 0.1 µg/mL and was selected for in vitro studies on isolated rat corpus cavernosum smooth muscle and in vivo sexual behaviour studies on healthy and diabetic rats. Rats were divided into five groups, each containing six animals and treated orally with either water, vehicle (1% Tween-20), MEMO (45 and 90 mg/kg/day for 21 days), and standard drug, sildenafil (5 mg/kg/day for 7 days). An equal number of female rats were used, and the effect of MEMO and sildenafil was compared with that of vehicle. MEMO significantly relaxed isolated rat corpus cavernosum smooth muscle at 0.1-100 µg/mL in vitro and significantly increased (p < 0.05) sexual activity, intracavernous pressure/mean arterial pressure in normal and diabetic rats. The increase in erectile function of rats by MEMO could be because of its sEH inhibitory activity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Use of a soluble epoxide hydrolase inhibitor in smoke-induced chronic obstructive pulmonary disease.

    PubMed

    Wang, Lei; Yang, Jun; Guo, Lei; Uyeminami, Dale; Dong, Hua; Hammock, Bruce D; Pinkerton, Kent E

    2012-05-01

    Tobacco smoke-induced chronic obstructive pulmonary disease (COPD) is a prolonged inflammatory condition of the lungs characterized by progressive and largely irreversible airflow limitation attributable to a number of pathologic mechanisms, including bronchitis, bronchiolitis, emphysema, mucus plugging, pulmonary hypertension, and small-airway obstruction. Soluble epoxide hydrolase inhibitors (sEHIs) demonstrated anti-inflammatory properties in a rat model after acute exposure to tobacco smoke. We compared the efficacy of sEHI t-TUCB (trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid) and the phosphodiesterase-4 (PDE4) inhibitor Rolipram (Biomol International, Enzo Life Sciences, Farmingdale, NY) to reduce lung injury and inflammation after subacute exposure to tobacco smoke over a period of 4 weeks. Pulmonary physiology, bronchoalveolar lavage, cytokine production, and histopathology were analyzed to determine the efficacy of sEHI and Rolipram to ameliorate tobacco smoke-induced inflammation and injury in the spontaneously hypertensive rat. Both t-TUCB and Rolipram inhibited neutrophil elevation in bronchoalveolar lavage. sEHI t-TUCB suppressed IFN-γ, while improving lung function by reducing tobacco smoke-induced total respiratory resistance and tissue damping (small-airway and peripheral tissue resistance). Increases in tobacco smoke-induced alveolar airspace size were attenuated by t-TUCB. Rolipram inhibited the production of airway mucus. Both t-TUCB and Rolipram inhibited vascular remodeling-related growth factor. These findings suggest that sEHI t-TUCB has therapeutic potential for treating COPD by improving lung function and attenuating the lung inflammation and emphysematous changes caused by tobacco smoke. To the best of our knowledge, this is the first report to demonstrate that sEHI exerts significant protective effects after repeated, subacute tobacco smoke-induced lung injury in a rat model of COPD.

  4. Use of a Soluble Epoxide Hydrolase Inhibitor in Smoke-Induced Chronic Obstructive Pulmonary Disease

    PubMed Central

    Wang, Lei; Yang, Jun; Guo, Lei; Uyeminami, Dale; Dong, Hua; Hammock, Bruce D.

    2012-01-01

    Tobacco smoke-induced chronic obstructive pulmonary disease (COPD) is a prolonged inflammatory condition of the lungs characterized by progressive and largely irreversible airflow limitation attributable to a number of pathologic mechanisms, including bronchitis, bronchiolitis, emphysema, mucus plugging, pulmonary hypertension, and small-airway obstruction. Soluble epoxide hydrolase inhibitors (sEHIs) demonstrated anti-inflammatory properties in a rat model after acute exposure to tobacco smoke. We compared the efficacy of sEHI t-TUCB (trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid) and the phosphodiesterase-4 (PDE4) inhibitor Rolipram (Biomol International, Enzo Life Sciences, Farmingdale, NY) to reduce lung injury and inflammation after subacute exposure to tobacco smoke over a period of 4 weeks. Pulmonary physiology, bronchoalveolar lavage, cytokine production, and histopathology were analyzed to determine the efficacy of sEHI and Rolipram to ameliorate tobacco smoke–induced inflammation and injury in the spontaneously hypertensive rat. Both t-TUCB and Rolipram inhibited neutrophil elevation in bronchoalveolar lavage. sEHI t-TUCB suppressed IFN-γ, while improving lung function by reducing tobacco smoke–induced total respiratory resistance and tissue damping (small-airway and peripheral tissue resistance). Increases in tobacco smoke–induced alveolar airspace size were attenuated by t-TUCB. Rolipram inhibited the production of airway mucus. Both t-TUCB and Rolipram inhibited vascular remodeling–related growth factor. These findings suggest that sEHI t-TUCB has therapeutic potential for treating COPD by improving lung function and attenuating the lung inflammation and emphysematous changes caused by tobacco smoke. To the best of our knowledge, this is the first report to demonstrate that sEHI exerts significant protective effects after repeated, subacute tobacco smoke–induced lung injury in a rat model of COPD. PMID

  5. Dysregulation of Soluble Epoxide Hydrolase and Lipidomic Profiles in Anorexia Nervosa

    PubMed Central

    Shih, Pei-an Betty; Yang, Jun; Morisseau, Christophe; German, J. Bruce; Van Zeeland, Ashley; Armando, Aaron M.; Quehenberger, Oswald; Bergen, Andrew W.; Magistretti, Pierre; Berrettini, Wade; Halmi, Katherine Ann; Schork, Nicholas; Hammock, Bruce D.; Kaye, Walter

    2015-01-01

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. AN tend to have an aversion to foods rich in fat. Because Epoxide Hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN, and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid, and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared to controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment. PMID:25824304

  6. Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa.

    PubMed

    Shih, P B; Yang, J; Morisseau, C; German, J B; Zeeland, A A Scott-Van; Armando, A M; Quehenberger, O; Bergen, A W; Magistretti, P; Berrettini, W; Halmi, K A; Schork, N; Hammock, B D; Kaye, W

    2016-04-01

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

  7. Involvement of cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer.

    PubMed Central

    Guengerich, F P; Johnson, W W; Ueng, Y F; Yamazaki, H; Shimada, T

    1996-01-01

    In recent years there has been considerable interest in the effect of variations in activities of xenobiotic-metabolizing enzymes on cancer incidence. This interest has accelerated with the development of methods for analyzing genetic polymorphisms. However, progress in epidemiology has been slow and the contributions of polymorphisms to risks from individual chemicals and mixtures are often controversial. A series of studies is presented to show the complexities encountered with a single chemical, aflatoxin B1 (AFB1). AFB1 is oxidized by human cytochrome P450 enzymes to several products. Only one of these, the 8,9-exo-epoxide, appears to be mutagenic and the others are detoxication products. P450 3A4, which can both activate and detoxicate AFB1, is found in the liver and the small intestine. In the small intestine, the first contact after oral exposure, epoxidation would not lead to liver cancer. The (nonenzymatic) half-life of the epoxide has been determined to be approximately 1 sec at 23 degrees C and neutral pH. Although the half-life is short, AFB1-8,9-exo-epoxide does react with DNA and glutathione S-transferase. Levels of these conjugates have been measured and combined with the rate of hydrolysis in a kinetic model to predict constants for binding of the epoxide with DNA and glutathione S-transferase. A role for epoxide hydrolase in alteration of AFB1 hepatocarcinogenesis has been proposed, although experimental evidence is lacking. Some inhibition of microsome-generated genotoxicity was observed with rat epoxide hydrolase; further information on the extent of contribution of this enzyme to AFB1 metabolism is not yet available. PMID:8781383

  8. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase.

    PubMed

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-08-25

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor-c-Src-increased Tyr phosphorylation of sEH and formation of an sEH-Akt-AMPK-eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt-AMPK-eNOS signaling cascade.

  9. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-01-01

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor–c-Src–increased Tyr phosphorylation of sEH and formation of an sEH–Akt–AMPK–eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt–AMPK–eNOS signaling cascade. PMID:26304753

  10. A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization.

    PubMed

    Beloti, Lilian L; Costa, Bruna Z; Toledo, Marcelo A S; Santos, Clelton A; Crucello, Aline; Fávaro, Marianna T P; Santiago, André S; Mendes, Juliano S; Marsaioli, Anita J; Souza, Anete P

    2013-10-01

    A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/β hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495μM and 0.24μM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.

  11. Enhancement of antinociception by coadministration of nonsteroidal anti-inflammatory drugs and soluble epoxide hydrolase inhibitors

    PubMed Central

    Schmelzer, Kara R.; Inceoglu, Bora; Kubala, Lukas; Kim, In-Hae; Jinks, Steven L.; Eiserich, Jason P.; Hammock, Bruce D.

    2006-01-01

    Combination therapies have long been used to treat inflammation while reducing side effects. The present study was designed to evaluate the therapeutic potential of combination treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) and previously undescribed soluble epoxide hydrolase inhibitors (sEHIs) in lipopolysaccharide (LPS)-challenged mice. NSAIDs inhibit cyclooxygenase (COX) enzymes and thereby decrease production of metabolites that lead to pain and inflammation. The sEHIs, such as 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), stabilize anti-inflammatory epoxy-eicosatrienoic acids, which indirectly reduce the expression of COX-2 protein. Here we demonstrate that the combination therapy of NSAIDs and sEHIs produces significantly beneficial effects that are additive for alleviating pain and enhanced effects in reducing COX-2 protein expression and shifting oxylipin metabolomic profiles. When administered alone, AUDA-BE decreased protein expression of COX-2 to 73 ± 6% of control mice treated with LPS only without altering COX-1 expression and decreased PGE2 levels to 52 ± 8% compared with LPS-treated mice not receiving any therapeutic intervention. When AUDA-BE was used in combination with low doses of indomethacin, celecoxib, or rofecoxib, PGE2 concentrations dropped to 51 ± 7, 84 ± 9, and 91 ± 8%, respectively, versus LPS control, without disrupting prostacyclin and thromboxane levels. These data suggest that these drug combinations (NSAIDs and sEHIs) produce a valuable beneficial analgesic and anti-inflammatory effect while prospectively decreasing side effects such as cardiovascular toxicity. PMID:16950874

  12. Production of monospecific antiserum to a cytosolic epoxide hydrolase from human liver.

    PubMed

    Qato, M K; Reinmund, S G; Guenthner, T M

    1990-01-15

    A method for the purification to apparent homogeneity of cytosolic trans-stilbene oxide hydrolase from human liver is presented. The method employed ion exchange and gel filtration chromatography. From 50 g of human liver, 4.9 mg of homogenous enzyme protein was obtained. Although the enzyme had lost much of its catalytic activity during purification, it was nevertheless suitable for the preparation of antibodies to the enzyme. Only one immunogenic species was present in the antigen preparation, but some antibodies that were cross-reactive to sites on catalase were present in the antiserum. These catalase-specific antibodies were removed by immunoaffinity chromatography, and an IgG fraction that is monospecific to the cytosolic epoxide hydrolase was obtained. The usefulness of antibodies to this enzyme in immunoblotting experiments, following either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focussing, as well as in enzyme-linked immunosorbent assays, is demonstrated.

  13. Heavy Chain Single Domain Antibodies to Detect Native Human Soluble Epoxide Hydrolase

    PubMed Central

    Cui, Yongliang; Li, Dongyang; Morisseau, Christophe; Yang, Jun; Wan, Debin; Rossotti, Martín A.; Gee, Shirley J.; González-Sapienza, Gualberto G.; Hammock, Bruce D.

    2015-01-01

    The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer and other diseases. However, there is not a simple, inexpensive and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal-variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the ELISA and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney and lung). The VHH based ELISA appears to be a new reliable method for monitoring the sEH, and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research. PMID:26229025

  14. Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6 alpha-oxide and certain xenobiotic alkene and arene oxides.

    PubMed

    Levin, W; Michaud, D P; Thomas, P E; Jerina, D M

    1983-02-01

    Metabolism of cholesterol 5,6 alpha-oxide to the 5,6-glycol is catalyzed by a rat liver microsomal epoxide hydrolase that is distinct from the microsomal epoxide hydrolase that metabolizes a wide range of xenobiotic alkene and arene oxides. The two enzymes are antigenically distinct, and the purified microsomal epoxide hydrolase that metabolizes xenobiotic oxides does not catalyze the hydration of cholesterol 5,6 alpha-oxide. In vivo treatment of rats with inducers of microsomal epoxide hydrolase does not enhance the activity of cholesterol 5,6 alpha-oxide hydrolase and, in some cases, actually depresses enzyme activity in the resultant microsomal preparations. Octene 1,2-oxide and benz[a]anthracene 5,6-oxide, both good substrates for xenobiotic epoxide hydrolase, are not competitive inhibitors of cholesterol oxide hydration by rat liver microsomes. The above results establish the existence of a liver microsomal epoxide hydrolase that is under different regulatory control and that appears to have a different substrate specificity than the well-characterized microsomal epoxide hydrolase involved in the metabolism of a widely diverse group of alkene and arene oxides.

  15. Efficient kinetic resolution of phenyl glycidyl ether by a novel epoxide hydrolase from Tsukamurella paurometabola.

    PubMed

    Wu, Kai; Wang, Hualei; Sun, Huihui; Wei, Dongzhi

    2015-11-01

    Enantioselective hydrolysis of racemic epoxides mediated by epoxide hydrolases (EHs) is one of the most promising approaches to obtain enantiopure epoxides. In this study, we identified and characterized a novel EH (TpEH1) from Tsukamurella paurometabola by analyzing the conserved catalytic residues of EH. TpEH1 was overexpressed and purified, and its catalytic properties were studied using racemic phenyl glycidyl ether (PGE) and its derivatives as substrates. TpEH1 showed excellent enantioselectivity to the substrates PGE, 3-methylPGE, and 3-nitroPGE. The highest enantioselectivity (E > 100) was achieved when 3-nitroPGE was used as the substrate. The recombinant Escherichia coli TpEH1 demonstrated high substrate tolerance toward PGE and could hydrolyze PGE at concentrations of up to 400 mM (60 g/L) with high enantioselectivity (E = 65), giving (R)-PGE with enantiomeric excess of more than 99 % ee and 45 % yield within 1 h. This concentration of PGE is the highest reported concentration catalyzed by native EHs to date. Thus, the easily available and highly active E. coli TpEH1 showed great potential for the practical preparation of optically pure (R)-PGE.

  16. Cloning and characterization of three epoxide hydrolases from a marine bacterium, Erythrobacter litoralis HTCC2594.

    PubMed

    Woo, Jung-Hee; Hwang, Young-Ok; Kang, Sung Gyun; Lee, Hyun Sook; Cho, Jang-Cheon; Kim, Sang-Jin

    2007-08-01

    Previously, we reported that ten strains belonging to Erythrobacter showed epoxide hydrolase (EHase) activities toward various epoxide substrates. Three genes encoding putative EHases were identified by analyzing open reading frames of Erythrobacter litoralis HTCC2594. Despite low similarities to reported EHases, the phylogenetic analysis of the three genes showed that eeh1 was similar to microsomal EHase, while eeh2 and eeh3 could be grouped with soluble EHases. The three EHase genes were cloned, and the recombinant proteins (rEEH1, rEEH2, and rEEH3) were purified. The functionality of purified proteins was proved by hydrolytic activities toward styrene oxide. EEH1 preferentially hydrolyzed (R)-styrene oxide, whereas EEH3 preferred to hydrolyze (S)-styrene oxide, representing enantioselective hydrolysis of styrene oxide. On the other hand, EEH2 could hydrolyze (R)- and (S)-styrene oxide at an equal rate. The optimal pH and temperature for the EHases occurred largely at neutral pHs and 40-55 degrees C. The substrate selectivity of rEEH1, rEEH2, and rEEH3 toward various epoxide substrates were also investigated. This is the first representation that a strict marine microorganism possessed three EHases with different enantioselectivity toward styrene oxide.

  17. Role of microsomal epoxide hydrolase in methamphetamine-induced drug dependence in mice.

    PubMed

    Shin, Eun-Joo; Bing, Guoying; Chae, Jong Seok; Kim, Tae Woo; Bach, Jae-Hyung; Park, Dae Hun; Yamada, Kiyofumi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2009-12-01

    Microsomal epoxide hydrolase (mEH) and cytochrome P-450 (CYP) ensure the rapid detoxification of epoxides generated during the oxidative metabolism of xenobiotics. Although CYP has been demonstrated to modulate methamphetamine (METH)-induced behavioral effects, little is known about the role of the mEH gene on these effects. We examined the role of mEH gene expression in METH-induced conditioned place preference and behavioral sensitization by using mEH(-/-) and wild-type (WT) mice. Extracellular dopamine (DA) levels and DA uptake into synaptosomes were assessed by using an in vivo microdialysis and [(3)H]DA uptake assay. We applied double-label immunocytochemistry to characterize mEH-positive cellular types. METH-induced behavioral responses paralleled striatal c-Fos-like immunoreactivity. METH treatment resulted in increased extracellular DA levels in the nucleus accumbens but decreased synaptosomal DA uptake in the striatum. These behavioral and neurochemical changes were more pronounced in the mEH(-/-) mice than in WT mice. In WT mice, mEH-like immunoreactivity was expressed in astrocytes labeled by GFAP or S100B after METH treatment. The results suggest that epoxide intermediates mediate METH drug dependence and that astrocytic reactions of mEH protein are important in the endogenous modulation in response to METH drug dependence. Copyright 2009 Wiley-Liss, Inc.

  18. The Molecular Structure of Epoxide Hydrolase B From And Its Complex With Urea-Based Inhibitor

    SciTech Connect

    Biswal, B.K.; Morisseau, C.; Garen, G.; Cherney, M.M.; Garen, C.; Niu, C.; Hammock, B.D.; James, M.N.G.

    2009-05-11

    Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC(50) approximately 19 nM) urea-based inhibitor at 2.1 and 2.4 A resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical alpha/beta hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the binding-site residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120 degrees about its C(alpha)-C(beta) bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the alpha/beta type.

  19. Bioactive lipid profiling reveals drug target engagement of a soluble epoxide hydrolase inhibitor in a murine model of tobacco smoke exposure.

    PubMed

    Nording, Malin L; Yang, Jun; Hoang, Laura; Zamora, Vanessa; Uyeminami, Dale; Espiritu, Imelda; Pinkerton, Kent E; Hammock, Bruce D; Luria, Ayala

    2015-04-01

    The inflammatory process underlying chronic obstructive pulmonary disease (COPD) may be caused by tobacco smoke (TS) exposure. Previous studies show that epoxyeicosatrienoic acids (EETs) possess promising anti-inflammatory properties, therefore stabilization of EETs and other fatty acid epoxides through inhibition of soluble epoxide hydrolase (sEH) was investigated in mouse models of acute and sub-chronic inflammation caused by TS exposure. During the entire TS exposure, the potent sEH inhibitor 1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was given via drinking water. To assess drug target engagement of TUPS, a tandem mass spectrometry method was used for bioactive lipid profiling of a broad range of fatty acid metabolites, including EETs, and their corresponding diols (DHETs) derived from arachidonic acid, as well as epoxides and diols derived from other fatty acids. Several, but not all, plasma epoxide/diol ratios increased in mice treated with sEH inhibitor, compared to non-treated mice suggesting a wider role for sEH involving more fatty acid precursors besides arachidonic acid. This study supports qualitative use of epoxide/diol ratios explored by bioactive lipid profiling to indicate drug target engagement in mouse models of TS exposure relevant to COPD, which may have ramifications for future therapeutic interventions of sEH.

  20. An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site.

    PubMed

    Abdel-Latief, Mohatmed; Garbe, Leif A; Koch, Markus; Ruther, Joachim

    2008-07-01

    Epoxide hydrolases (EHs) are enzymes occurring in virtually any living organism. They catalyze the hydrolysis of epoxide containing lipids and are involved in crucial mechanisms, such as the detoxification of xenobiotics or the regulation of inflammation and blood pressure. Here, we describe a function of a putative EH gene in the biosynthesis of a sex attractant in the jewel wasp Nasonia vitripennis and use this gene to localize the site of pheromone production. Males of this parasitic wasp release a mixture of (4R,5R)-( threo-) and (4R,5S)-( erythro-)5-hydroxy-4-decanolide (HDL) to attract virgin females. Using a stable isotope labeled precursor, we demonstrated that vernolic acid ( erythro-12,13-epoxy-octadec-9Z-enoic acid) is converted by N. vitripennis males to threo-HDL. This suggested the involvement of an EH in hydrolyzing the fatty acid epoxide under inversion of the stereochemistry into the respective diol, which might be further processed by chain shortening and lactonization to HDL. We cloned a putative N. vitripennis EH gene (Nasvi-EH1) encoding 470 amino acids and localized its transcripts in the male rectal papillae by in situ RT-PCR. Chemical analyses and histological studies confirmed that males synthesize the sex attractant in the rectal vesicle and release it via the anal orifice. Involvement of Nasvi-EH1 in HDL biosynthesis was established by RNAi-mediated gene silencing. Injection of Nasvi-EH1 dsRNA into male abdomens inhibited pheromone biosynthesis by 55% and suppressed the targeted gene transcripts in the rectal vesicle by 95%.

  1. An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site

    PubMed Central

    Abdel-latief, Mohatmed; Garbe, Leif A.; Koch, Markus; Ruther, Joachim

    2008-01-01

    Epoxide hydrolases (EHs) are enzymes occurring in virtually any living organism. They catalyze the hydrolysis of epoxide containing lipids and are involved in crucial mechanisms, such as the detoxification of xenobiotics or the regulation of inflammation and blood pressure. Here, we describe a function of a putative EH gene in the biosynthesis of a sex attractant in the jewel wasp Nasonia vitripennis and use this gene to localize the site of pheromone production. Males of this parasitic wasp release a mixture of (4R,5R)-(threo-) and (4R,5S)-(erythro-)5-hydroxy-4-decanolide (HDL) to attract virgin females. Using a stable isotope labeled precursor, we demonstrated that vernolic acid (erythro-12,13-epoxy-octadec-9Z-enoic acid) is converted by N. vitripennis males to threo-HDL. This suggested the involvement of an EH in hydrolyzing the fatty acid epoxide under inversion of the stereochemistry into the respective diol, which might be further processed by chain shortening and lactonization to HDL. We cloned a putative N. vitripennis EH gene (Nasvi-EH1) encoding 470 amino acids and localized its transcripts in the male rectal papillae by in situ RT-PCR. Chemical analyses and histological studies confirmed that males synthesize the sex attractant in the rectal vesicle and release it via the anal orifice. Involvement of Nasvi-EH1 in HDL biosynthesis was established by RNAi-mediated gene silencing. Injection of Nasvi-EH1 dsRNA into male abdomens inhibited pheromone biosynthesis by 55% and suppressed the targeted gene transcripts in the rectal vesicle by 95%. PMID:18579785

  2. Design and synthesis of dual modulators of soluble epoxide hydrolase and peroxisome proliferator-activated receptors.

    PubMed

    la Buscató, Estel; Blöcher, René; Lamers, Christina; Klingler, Franca-Maria; Hahn, Steffen; Steinhilber, Dieter; Schubert-Zsilavecz, Manfred; Proschak, Ewgenij

    2012-12-13

    Metabolic syndrome is a complex condition which often requires the use of multiple medications as a treatment. The resulting problems of polypharmacy are increase in side effects, drug-drug interactions, and its high economic cost. Development of multitarget compounds is a promising strategy to avoid the complications arising from administration of multiple drugs. Modulators of peroxisome proliferator-activated receptors (PPARs) are established agents in the treatment of dyslipidaemia, hyperglycaemia, and insulin resistance. Inhibitors of soluble epoxide hydrolase (sEH) are under evaluation for their use in cardiovascular diseases. In the present study, a series of dual sEH/PPAR modulators containing a pyrrole acidic headgroup and a urea pharmacophore were designed, synthesized, and evaluated in vitro using recombinant enzyme and cell-based assays. Compounds with different activity profiles were obtained which could be used in the treatment of metabolic syndrome.

  3. Soluble epoxide hydrolase inhibitor 1-trifluoromethoxyphenyl-3- (1-propionylpiperidin-4-yl) urea attenuates bleomycin-induced pulmonary fibrosis in mice.

    PubMed

    Zhou, Yong; Yang, Jun; Sun, Guo-Ying; Liu, Tian; Duan, Jia-Xi; Zhou, Hui-Fang; Lee, Kin Sing; Hammock, Bruce D; Fang, Xiang; Jiang, Jian-Xin; Guan, Cha-Xiang

    2016-02-01

    Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties and inhibition of sEH might provide protective effects against inflammatory fibrosis. We test the effects of a selected sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), on bleomycin-induced pulmonary fibrosis (PF) in mice. A mouse model of PF was established by intratracheal injection of bleomycin and TPPU was administered for 21 days after bleomycin injection. We found TPPU treatment improved the body weight loss and survival rate of bleomycin-stimulated mice. Histological examination showed that TPPU treatment alleviated bleomycin-induced inflammation and maintained the alveolar structure of the pulmonary tissues. TPPU also decreased the bleomycin-induced deposition of collagen and the expression of procollagen I mRNA in lung tissues of mice. TPPU decreased the transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β) and IL-6 levels in the serum of bleomycin-stimulated mice. Furthermore, TPPU inhibited the proliferation and collagen synthesis of mouse fibroblasts and partially reversed TGF-β1-induced α-smooth muscle actin expression. Our results indicate that the inhibition of sEH attenuates bleomycin-induced inflammation and collagen deposition and therefore prevents bleomycin-induced PF in a mouse model.

  4. Soluble epoxide hydrolase inhibitor 1-trifluoromethoxyphenyl-3- (1-propionylpiperidin-4-yl) urea attenuates bleomycin-induced pulmonary fibrosis in mice

    PubMed Central

    Zhou, Yong; Sun, Guo-Ying; Liu, Tian; Duan, Jia-Xi; Zhou, Hui-Fang; Lee, Kin Sing; Hammock, Bruce D.; Fang, Xiang; Guan, Cha-Xiang; Jiang, Jian-Xin

    2015-01-01

    Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs, but not their diols, have anti-inflammatory properties, and inhibition of sEH might provide protective effects against inflammatory fibrosis. In this study, we tested the effects of a selected sEH inhibitor, 1-trifluoromethoxyphenyl-3- (1-propionylpiperidin-4-yl) urea (TPPU), on bleomycin-induced pulmonary fibrosis (PF) in mice. A mouse model of PF was established by intratracheal injection of bleomycin, and TPPU was administered for 21 days after bleomycin injection. We found TPPU treatment improved the body weight loss and survival rate of bleomycin-stimulated mice. Histological examinations showed that TPPU treatment alleviated bleomycin-induced inflammation, and maintained alveolar structure of pulmonary tissues. TPPU also decreased bleomycin-induced deposition of collagen, and expression of the procollagen I mRNA in lung tissues of mice. TPPU decreased the TGF-β1, IL-1β and IL-6 levels in serum of bleomycin-stimulated mice. Moreover, TPPU inhibited proliferation, collagen synthesis of the mouse fibroblasts, and partially reversed TGF-β1-induced α-SMA expression. Our results indicated that inhibition of sEH attenuates bleomycin-induced inflammation, collagen deposition, and therefore prevents bleomycin-induced PF in mice model. PMID:26310139

  5. An Orally Active Epoxide Hydrolase Inhibitor Lowers Blood Pressure and Provides Renal Protection in Salt-Sensitive Hypertension

    PubMed Central

    Imig, John D.; Zhao, Xueying; Zaharis, Constantine Z.; Olearczyk, Jeffrey J.; Pollock, David M.; Newman, John W.; Kim, In-Hae; Watanabe, Takaho; Hammock, Bruce D.

    2006-01-01

    The present study tested the hypothesis that increasing epoxyeicosatrienoic acids by inhibition of soluble epoxide hydrolase (sEH) would lower blood pressure and ameliorate renal damage in salt-sensitive hypertension. Rats were infused with angiotensin and fed a normal-salt diet or an 8% NaCl diet for 14 days. The sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), was given orally to angiotensin-infused animals during the 14-day period. Plasma AUDA metabolite levels were measured, and they averaged 10±2 ng/mL in normal-salt angiotensin hypertension and 19±3 ng/mL in high-salt angiotensin hypertension on day 14 in the animals administered the sEH inhibitor. Mean arterial blood pressure averaged 161±4 mm Hg in normal-salt and 172±5 mmHg in the high-salt angiotensin hypertension groups on day 14. EH inhibitor treatment significantly lowered blood pressure to 140±5 mm Hg in the normal-salt angiotensin hypertension group and to 151±6 mm Hg in the high-salt angiotensin hypertension group on day 14. The lower arterial blood pressures in the AUDA-treated groups were associated with increased urinary epoxide-to-diol ratios. Urinary microalbumin levels were measured, and ED-1 staining was used to determine renal damage and macrophage infiltration in the groups. Two weeks of AUDA treatment decreased urinary microalbumin excretion in the normal-salt and high-salt angiotensin hypertension groups and macrophage number in the high-salt angiotensin hypertension group. These data demonstrate that sEH inhibition lowers blood pressure and ameliorates renal damage in angiotensin-dependent, salt-sensitive hypertension. PMID:16157792

  6. Epoxide hydrolase: a mRNA induced by the fungal pathogen Alternaria alternata on rough lemon (Citrus jambhiri Lush).

    PubMed

    Gomi, Kenji; Yamamato, Hiroyuki; Akimitsu, Kazuya

    2003-09-01

    An expression profile of genes induced by non-pathogenic Alternaria alternata on rough lemon leaves was obtained by sequencing 500 subtractive PCR clones generated from mRNA of leaves inoculated with the fungus after subtraction with that of non-inoculated leaves. About 6% of the cDNA sequences had homology to known putative defense-related genes including epoxide hydrolase. A full-length cDNA (951 bp) from rough lemon that encoded epoxy hydrolase was isolated by random amplification of cDNA ends (RACEs), based on sequence information from subtractive PCR, and designated as RlemEH. The product of this gene expressed with an in vitro translation system with Escherichia coli also had activity of a soluble type of epoxide hydrolase. The transcript of rough lemon RlemEH was not detected in flowers, fruits, stems or leaves, but was induced after inoculation of leaves with conidia of Alternaria alternata, wounding, or treatment with C6 volatiles, including trans-2-hexenol and cis-3-hexenol, and methyl jasmonate. The response of the epoxide hydrolase gene correlated well with the activation of defense mechanisms induced in plant-fungus interactions.

  7. Soluble epoxide hydrolase deficiency alters pancreatic islet size and improves glucose homeostasis in a model of insulin resistance.

    PubMed

    Luria, Ayala; Bettaieb, Ahmed; Xi, Yannan; Shieh, Guang-Jong; Liu, Hsin-Chen; Inoue, Hiromi; Tsai, Hsing-Ju; Imig, John D; Haj, Fawaz G; Hammock, Bruce D

    2011-05-31

    Visceral obesity has been defined as an important element of the metabolic syndrome and contributes to the development of insulin resistance and cardiovascular disease. Increasing endogenous levels of epoxyeicosatrienoic acids (EETs) are known for their analgesic, antihypertensive, and antiinflammatory effects. The availability of EETs is limited primarily by the soluble epoxide hydrolase (sEH, EPHX2), which metabolizes EETs to their less active diols. In this study, we tested the hypothesis that EETs are involved in glucose regulation and in retarding the development of insulin resistance. To address the role of EETs in regulating glucose homeostasis and insulin signaling, we used mice with targeted gene deletion of sEH (Ephx2-null mice) and a subsequent study with a selective sEH inhibitor. When wild-type mice are fed a high fat diet, insulin resistance develops. However, knockout or inhibition of sEH activity resulted in a significant decrease in plasma glucose. These findings are characterized by enhancement of tyrosyl phosphorylation of the insulin receptor, insulin receptor substrate 1, and their downstream cascade. In addition, pancreatic islets were larger when sEH was disrupted. This effect was associated with an increase in vasculature. These observations were supported by pharmacological inhibition of sEH. These data suggest that an increase in EETs due to sEH-gene knockout leads to an increase in the size of islets and improved insulin signaling and sensitivity.

  8. Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    PubMed Central

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  9. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

    PubMed

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes.

  10. Structural insights into human microsomal epoxide hydrolase by combined homology modeling, molecular dynamics simulations, and molecular docking calculations.

    PubMed

    Saenz-Méndez, Patricia; Katz, Aline; Pérez-Kempner, María Lucía; Ventura, Oscar N; Vázquez, Marta

    2017-04-01

    A new homology model of human microsomal epoxide hydrolase was derived based on multiple templates. The model obtained was fully evaluated, including MD simulations and ensemble-based docking, showing that the quality of the structure is better than that of only previously known model. Particularly, a catalytic triad was clearly identified, in agreement with the experimental information available. Analysis of intermediates in the enzymatic mechanism led to the identification of key residues for substrate binding, stereoselectivity, and intermediate stabilization during the reaction. In particular, we have confirmed the role of the oxyanion hole and the conserved motif (HGXP) in epoxide hydrolases, in excellent agreement with known experimental and computational data on similar systems. The model obtained is the first one that fully agrees with all the experimental observations on the system. Proteins 2017; 85:720-730. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  11. Effect of Soluble Epoxide Hydrolase on the Modulation of Coronary Reactive Hyperemia: Role of Oxylipins and PPARγ

    PubMed Central

    Hanif, Ahmad; Edin, Matthew L.; Zeldin, Darryl C.; Morisseau, Christophe; Nayeem, Mohammed A.

    2016-01-01

    Coronary reactive hyperemia (CRH) is a physiological response to ischemic insult that prevents the potential harm associated with an interruption of blood supply. The relationship between the pharmacologic inhibition of soluble epoxide hydrolase (sEH) and CRH response to a brief ischemia is not known. sEH is involved in the main catabolic pathway of epoxyeicosatrienoic acids (EETs), which are converted into dihydroxyeicosatrienoic acids (DHETs). EETs protect against ischemia/reperfusion injury and have numerous beneficial physiological effects. We hypothesized that inhibition of sEH by t-AUCB enhances CRH in isolated mouse hearts through changing the oxylipin profiles, including an increase in EETs/DHETs ratio. Compared to controls, t-AUCB–treated mice had increased CRH, including repayment volume (RV), repayment duration, and repayment/debt ratio (p < 0.05). Treatment with t-AUCB significantly changed oxylipin profiles, including an increase in EET/DHET ratio, increase in EpOME/DiHOME ratio, increase in the levels of HODEs, decrease in the levels of mid-chain HETEs, and decrease in prostanoids (p < 0.05). Treatment with MS-PPOH (CYP epoxygenase inhibitor) reduced CRH, including RV (p < 0.05). Involvement of PPARγ in the modulation of CRH was demonstrated using a PPARγ-antagonist (T0070907) and a PPARγ-agonist (rosiglitazone). T0070907 reduced CRH (p < 0.05), whereas rosiglitazone enhanced CRH (p < 0.05) in isolated mouse hearts compared to the non-treated. These data demonstrate that sEH inhibition enhances, whereas CYP epoxygenases-inhibition attenuates CRH, PPARγ mediate CRH downstream of the CYP epoxygenases-EET pathway, and the changes in oxylipin profiles associated with sEH-inhibition collectively contributed to the enhanced CRH. PMID:27583776

  12. Soluble Epoxide Hydrolase Null Mice Exhibit Female and Male Differences In Regulation of Vascular Homeostasis

    PubMed Central

    Vanella, Luca; Canestraro, Martina; Lee, Craig R.; Cao, Jian; Zeldin, Darryl C.; Schwartzman, Michal L.; Abraham, Nader G.

    2015-01-01

    Increased CYP epoxygenase activity and consequently up regulation of epoxyeicosatrienoic acids (EETs) levels provides protection against metabolic syndrome and cardiovascular diseases. Conversion of arachidonic acid epoxides to diols by soluble epoxide hydrolase (sEH) diminishes the beneficial cardiovascular properties of these epoxyeicosanoids. We therefore examined the possible biochemical consequences of sEH deletion on vascular responses in male and female mice. Through the use of the sEH KO mouse, we provide evidence of differences in the compensatory response in the balance between nitric oxide (NO), carbon monoxide (CO), EETs and the vasoconstrictor 20-HETE in male and female KO mice. Serum levels of adiponectin, TNFα, IL-1b and MCP1 and protein expression in vascular tissue of p-AMPK, p-AKT and p-eNOS were measured. Deletion of sEH caused a significant (p<0,05) decrease in body weight, and an increase in adiponectin, pAMPK and pAKT levels in female KO mice compared to male KO mice. Gene deletion resulted in a higher production of renal EETs in female KO compared to male KO mice and, concomitantly, we observed an increase in renal 20-HETEs levels and superoxide anion production only in male KO mice. sEH deletion increased p-AKT and p-eNOS protein expression but decreased p-AMPK levels in female KO mice. Increased levels of p-eNOS at Thr-495 were observed only in KO male mice. While p-eNOS at 1177 were not significantly different between male and female. Nitric oxide production was unaltered in male KO mice. These results provide evidence of gender differences in the preservation of vascular homeostasis in response to sEH deletion which involves regulation of phosphorylation of eNOS at the 495 site. PMID:25908301

  13. Effect of a Soluble Epoxide Hydrolase Inhibitor, UC1728, on LPS-Induced Uveitis in the Rabbit

    PubMed Central

    McLellan, Gillian J.; Aktas, Zeynep; Hennes-Beean, Elizabeth; Kolb, Aaron W.; Larsen, Inna V.; Schmitz, Emily J.; Clausius, Hilary R.; Yang, Jun; Hwang, Sung Hee; Morisseau, Christophe; Inceoglu, Bora; Hammock, Bruce D.; Brandt, Curtis R.

    2016-01-01

    Cytochrome P450 epoxygenase isozymes convert free arachidonic acid into eicosanoids named epoxyeicosatrienoic acids (EETs) that have roles in regulating inflammation. EETs are rapidly converted to dihydroxyeicosatrienoic acids (DiHETs) by soluble epoxide hydrolase (sEH). Little is known about the potential role of these metabolites in uveitis, but conversion of EETs to DiHETs could contribute to the inflammation. We tested a potent and orally available inhibitor of sEH for its ability to reduce ocular inflammation in a rabbit LPS-induced model of uveitis. Rabbits were treated by subcutaneous injection with the sEH inhibitor (UC1728, 3 mg/kg), or the vehicle control (PEG400) and uveitis was assessed at 6, 24 and 48 h post-intracameral LPS injection using a modified Hackett-McDonald scoring system. Eyes treated by intra-cameral injection of PBS, or by aseptic preparation served as further controls. Signs of inflammation in this model were mild and transient. Treatment with UC1728 did not significantly reduce inflammation compared to animals treated with the PEG400 vehicle. Blood levels of UC1728 were a thousand fold higher than the in vitro determined inhibitory potency (IC50) of the compound suggesting a significant degree of inhibition of sEH in the rabbit. The lack of efficacy suggests that sEH or its substrates the EETs may not be involved in mediating inflammation in this model of uveitis. PMID:28066796

  14. Metabolic products of soluble epoxide hydrolase are essential for monocyte chemotaxis to MCP-1 in vitro and in vivo.

    PubMed

    Kundu, Suman; Roome, Talat; Bhattacharjee, Ashish; Carnevale, Kevin A; Yakubenko, Valentin P; Zhang, Renliang; Hwang, Sung Hee; Hammock, Bruce D; Cathcart, Martha K

    2013-02-01

    Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A(2) (cPLA(2)). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA(2)-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA(2) activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.

  15. Genetic analysis of microsomal epoxide hydrolase gene and its association with lung cancer risk

    PubMed Central

    Park, Jong Y.; Chen, Lan; Elahi, Abul; Lazarus, Philip; Tockman, Melvyn S.

    2013-01-01

    The human microsomal epoxide hydrolase (EH) gene contains polymorphic alleles, which may be linked to increased risk for tobacco-related lung cancer. The purpose of this study is to screen new polymorphisms and determine whether these polymorphisms can be used to predict individual susceptibility to lung cancer. The PCR-single strand conformation polymorphism (SSCP) analysis was used to screen for polymorphisms in the coding region of the EH gene. Eleven polymorphisms, including previously reported polymorphisms, were identified and the prevalence of these variants was assessed in at least 50 healthy Caucasians and African Americans. Among the eleven polymorphisms, the prevalence of the amino acid-changing EH polymorphisms in codons 43, 113, and 139 was examined in 182 Caucasian incident cases with primary lung cancer, as well as in 365 frequency-matched controls to examine the role of EH polymorphisms in lung cancer risk. A significant increase in lung cancer risk was observed for predicted high EH activity genotypes (OR = 2.3, 95% CI = 1.2–4.3) as compared to low EH activity genotypes. This association was more pronounced among patients with lung adenocarcinoma (OR = 4.7, 95% CI = 1.7–13.1). These results suggest that the EH polymorphism plays an important role in lung cancer risk and is linked to tobacco smoke exposure. PMID:15901990

  16. Role of soluble epoxide hydrolase in age-related vascular cognitive decline.

    PubMed

    Nelson, Jonathan W; Young, Jennifer M; Borkar, Rohan N; Woltjer, Randy L; Quinn, Joseph F; Silbert, Lisa C; Grafe, Marjorie R; Alkayed, Nabil J

    2014-10-01

    P450 eicosanoids are important regulators of the cerebral microcirculation, but their role in cerebral small vessel disease is unclear. We tested the hypothesis that vascular cognitive impairment (VCI) is linked to reduced cerebral microvascular eicosanoid signaling. We analyzed human brain tissue from individuals formerly enrolled in the Oregon Brain Aging Study, who had a history of cognitive impairment histopathological evidence of microvascular disease. VCI subjects had significantly higher lesion burden both on premortem MRI and postmortem histopathology compared to age- and sex-matched controls. Mass spectrometry-based eicosanoid analysis revealed that 14,15-dihydroxyeicosatrienoic acid (DHET) was elevated in cortical brain tissue from VCI subjects. Immunoreactivity of soluble epoxide hydrolase (sEH), the enzyme responsible for 14,15-DHET formation, was localized to cerebral microvascular endothelium, and was enhanced in microvessels of affected tissue. Finally, we evaluated the genotype frequency of two functional single nucleotide polymorphisms of sEH gene EPHX2 in VCI and control groups. Our findings support a role for sEH and a potential benefit from sEH inhibitors in age-related VCI. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Occurrence of urea-based soluble epoxide hydrolase inhibitors from the plants in the order Brassicales.

    PubMed

    Kitamura, Seiya; Morisseau, Christophe; Harris, Todd R; Inceoglu, Bora; Hammock, Bruce D

    2017-01-01

    Recently, dibenzylurea-based potent soluble epoxide hydrolase (sEH) inhibitors were identified in Pentadiplandra brazzeana, a plant in the order Brassicales. In an effort to generalize the concept, we hypothesized that plants that produce benzyl glucosinolates and corresponding isothiocyanates also produce these dibenzylurea derivatives. Our overall aim here was to examine the occurrence of urea derivatives in Brassicales, hoping to find biologically active urea derivatives from plants. First, plants in the order Brassicales were analyzed for the presence of 1, 3-dibenzylurea (compound 1), showing that three additional plants in the order Brassicales produce the urea derivatives. Based on the hypothesis, three dibenzylurea derivatives with sEH inhibitory activity were isolated from maca (Lepidium meyenii) roots. Topical application of one of the identified compounds (compound 3, human sEH IC50 = 222 nM) effectively reduced pain in rat inflammatory pain model, and this compound was bioavailable after oral administration in mice. The biosynthetic pathway of these urea derivatives was investigated using papaya (Carica papaya) seed as a model system. Finally, a small collection of plants from the Brassicales order was grown, collected, extracted and screened for sEH inhibitory activity. Results show that several plants of the Brassicales order could be potential sources of urea-based sEH inhibitors.

  18. Rational design of potent and selective inhibitors of an epoxide hydrolase virulence factor from Pseudomonas aeruginosa

    PubMed Central

    Kitamura, Seiya; Hvorecny, Kelli L.; Niu, Jun; Hammock, Bruce D.; Madden, Dean R.; Morisseau, Christophe

    2016-01-01

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. Based on these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif, and have the potential for clinical application. PMID:27120257

  19. Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

    PubMed

    Kitamura, Seiya; Hvorecny, Kelli L; Niu, Jun; Hammock, Bruce D; Madden, Dean R; Morisseau, Christophe

    2016-05-26

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.

  20. Occurrence of urea-based soluble epoxide hydrolase inhibitors from the plants in the order Brassicales

    PubMed Central

    Kitamura, Seiya; Morisseau, Christophe; Harris, Todd R.; Inceoglu, Bora

    2017-01-01

    Recently, dibenzylurea-based potent soluble epoxide hydrolase (sEH) inhibitors were identified in Pentadiplandra brazzeana, a plant in the order Brassicales. In an effort to generalize the concept, we hypothesized that plants that produce benzyl glucosinolates and corresponding isothiocyanates also produce these dibenzylurea derivatives. Our overall aim here was to examine the occurrence of urea derivatives in Brassicales, hoping to find biologically active urea derivatives from plants. First, plants in the order Brassicales were analyzed for the presence of 1, 3-dibenzylurea (compound 1), showing that three additional plants in the order Brassicales produce the urea derivatives. Based on the hypothesis, three dibenzylurea derivatives with sEH inhibitory activity were isolated from maca (Lepidium meyenii) roots. Topical application of one of the identified compounds (compound 3, human sEH IC50 = 222 nM) effectively reduced pain in rat inflammatory pain model, and this compound was bioavailable after oral administration in mice. The biosynthetic pathway of these urea derivatives was investigated using papaya (Carica papaya) seed as a model system. Finally, a small collection of plants from the Brassicales order was grown, collected, extracted and screened for sEH inhibitory activity. Results show that several plants of the Brassicales order could be potential sources of urea-based sEH inhibitors. PMID:28472063

  1. Induction of Epoxide Hydrolase, Glucuronosyl Transferase, and Sulfotransferase by Phenethyl Isothiocyanate in Male Wistar Albino Rats

    PubMed Central

    Mohd Noor, Noramaliza; Konsue, Nattaya

    2014-01-01

    Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention. PMID:24592387

  2. Ovarian expressed microsomal epoxide hydrolase: Role in detoxification of 4-vinylcyclohexene diepoxide and regulation by phosphatidylinositol-3 kinase signaling

    SciTech Connect

    Bhattacharya, Poulomi; Sen, Nivedita; Hoyer, Patricia B.; Keating, Aileen F.

    2012-01-01

    4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally, because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P < 0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased (P < 0.05) on day 4 of VCD (30 μM) exposure, followed by increased (P < 0.05) mEH protein after 6 days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH. -- Highlights: ► Ovarian mEH functions to metabolize VCD to a less toxic compound. ► mEH expression is increased in a temporal pattern in response to VCD exposure. ► PI3K signaling is involved in regulation of ovarian mEH expression.

  3. Soluble epoxide hydrolase activity regulates inflammatory responses and seizure generation in two mouse models of temporal lobe epilepsy.

    PubMed

    Hung, Yu-Wen; Hung, Shao-Wen; Wu, Yi-Chen; Wong, Lin-King; Lai, Ming-Tsong; Shih, Yang-Hsin; Lee, Tzong-Shyuan; Lin, Yung-Yang

    2015-01-01

    Neuroinflammation is known to be involved in epileptogenesis with unclear mechanisms. Inhibition of soluble epoxide hydrolase (sEH) seems to offer anti-inflammatory protection to ischemic brain injury in rodents. Thus, it is hypothesized that sEH inhibition might also affect the neuroinflammatory responses caused by epileptic seizures. In the present study, we investigated the involvement of sEH in neuroinflammation, seizure generation and subsequent epileptogenesis using two mouse models of temporal lobe epilepsy. Experimental epileptic seizures were induced by either pilocarpine or electrical amygdala kindling in both wild-type (WT) C57BL/6 mice and sEH knockout (sEH KO) mice. The sEH expression in the hippocampus was detected by immunohistochemistry and Western blot analysis. The effects of the sEH hydrolase inhibitors, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU), and of the genetic deletion of sEH on seizure-induced neuroinflammatory responses and the development of epilepsy were evaluated. In the hippocampus of WT mice, sEH was mainly expressed in astrocytes (GFAP(+)), neurons (NeuN(+)) and scattered microglia (Iba-1(+)) in the regions of CA1, CA3 and dentate gyrus. Expression of sEH was significantly increased on day 7, 14, 21 and 28 after pilocarpine-induced status epilepticus (SE). Administration with sEH inhibitors attenuated the SE-induced up-regulation of interleukin-1β (IL-1β) and interleukin-6 (IL-6), the degradation of EETs, as well as IκB phosphorylation. Following treatment with AUDA, the frequency and duration of spontaneous motor seizures in the pilocarpine-SE mice were decreased and the seizure-induction threshold of the fully kindled mice was increased. Up-regulation of hippocampal IL-1β and IL-6 was found in both WT and sEH KO mice after successful induction of SE. Notably, sEH KO mice were more susceptible to seizures than WT mice. Seizure related

  4. Effect of N-acetylcysteine in COPD patients with different microsomal epoxide hydrolase genotypes

    PubMed Central

    Zhang, Jian-Qing; Zhang, Jia-Qiang; Liu, Hua; Zhao, Zhi-Huan; Fang, Li-Zhou; Liu, Ling; Fu, Wei-Ping; Shu, Jing-Kui; Feng, Jia-Gang; Dai, Lu-Ming

    2015-01-01

    Background The role of the antioxidant N-acetylcysteine (NAC) in the treatment of chronic obstructive pulmonary disease (COPD) has not been clarified as yet. In early studies, we found that the proportion of smokers with COPD having extremely slow/slow microsomal epoxide hydrolase (EPHX1) enzyme activity is significantly higher than that in healthy smokers. The purpose of this study was to evaluate whether different EPHX1 enzyme activity is related to differential therapeutic effects of treatment with NAC in COPD. Methods A total of 219 patients with COPD were randomly allocated to an extremely slow/slow EPHX1 enzyme activity group (n=157) or a fast/normal EPHX1 enzyme activity group (n=62) according to their EPHX1 enzyme activity. Both groups were treated with NAC 600 mg twice daily for one year. The main study parameters, including forced expiratory volume in one second (FEV1), St George’s Respiratory Questionnaire (SGRQ), and yearly exacerbation rate, were measured at baseline and at 6-month intervals for one year. Results Both FEV1 and SGRQ symptom scores were improved after treatment with NAC in the slow activity group when compared with the fast activity group. Further, changes in FEV1 and SGRQ symptom score in patients with mild-to-moderate COPD were more significant than those in patients with severe-to-very severe COPD. The yearly exacerbation rates were reduced in both groups, but the reduction in the slow activity group was significantly lower than in the fast activity group. Conclusion NAC treatment in COPD patients with extremely slow/slow EPHX1 enzyme activity improves FEV1 and the SGRQ symptom score, especially in those with mild-to-moderate COPD, and polymorphism in the EPHX1 gene may have a significant role in differential responses to treatment with NAC in patients with COPD. PMID:25999707

  5. Colorectal polyp type and the association with charred meat consumption, smoking, and microsomal epoxide hydrolase polymorphisms

    PubMed Central

    Burnett-Hartman, Andrea N.; Newcomb, Polly A.; Mandelson, Margaret T.; Adams, Scott V.; Wernli, Karen J.; Shadman, Mazyar; Wurscher, Michelle A.; Makar, Karen W.

    2011-01-01

    Objective We determined the association between charred meat consumption, cigarette smoking, microsomal epoxide hydrolase (mEH) polymorphisms [rs1051740 and rs2234922], and colorectal adenomas and hyperplastic polyps (HPs) and explored gene-environment interactions. Methods Men and women with colorectal adenomas (n=519), HPs (n=691), or concurrently with both types of polyps (n=227) and polyp-free controls (n=772) receiving a colonoscopy from 12/04-9/07 were recruited. Participants completed telephone interviews and provided buccal cell samples; genotyping of mEH was completed using Taqman assays. We conducted polytomous regression and calculated odd ratios (OR) and 95% confidence intervals. Interactions were evaluated using Wald chi-square tests. Results Consumption of >3 servings of charred meat per week was associated with distal HPs (OR=2.0, 1.2–3.4) but not adenomas nor either type of proximal polyp. Heavy cigarette smoking (≥22 pack-years) was associated with an increased risk for colorectal adenomas (OR=1.7, 95% CI 1.2–2.4), HPs (OR=2.4, 95% CI 1.7–3.3), and both types (OR=2.8, 95% CI 1.8–4.3) with the strongest association for distal polyps. There was no association between mEH genotype and colorectal polyps, nor were any statistically significant gene-environment interactions identified. Discussion Future investigation of BaP exposure and colorectal neoplasia should analyze whether associations are dependent upon anatomic location. PMID:21598178

  6. Genetic enhancement of microsomal epoxide hydrolase improves metabolic detoxification but impairs cerebral blood flow regulation.

    PubMed

    Marowsky, Anne; Haenel, Karen; Bockamp, Ernesto; Heck, Rosario; Rutishauser, Sibylle; Mule, Nandkishor; Kindler, Diana; Rudin, Markus; Arand, Michael

    2016-12-01

    Microsomal epoxide hydrolase (mEH) is a detoxifying enzyme for xenobiotic compounds. Enzymatic activity of mEH can be greatly increased by a point mutation, leading to an E404D amino acid exchange in its catalytic triad. Surprisingly, this variant is not found in any vertebrate species, despite the obvious advantage of accelerated detoxification. We hypothesized that this evolutionary avoidance is due to the fact that the mEH plays a dualistic role in detoxification and control of endogenous vascular signaling molecules. To test this, we generated mEH E404D mice and assessed them for detoxification capacity and vascular dynamics. In liver microsomes from these mice, turnover of the xenobiotic compound phenanthrene-9,10-oxide was four times faster compared to WT liver microsomes, confirming accelerated detoxification. mEH E404D animals also showed faster metabolization of a specific class of endogenous eicosanoids, arachidonic acid-derived epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). Significantly higher DHETs/EETs ratios were found in mEH E404D liver, urine, plasma, brain and cerebral endothelial cells compared to WT controls, suggesting a broad impact of the mEH mutant on endogenous EETs metabolism. Because EETs are strong vasodilators in cerebral vasculature, hemodynamics were assessed in mEH E404D and WT cerebral cortex and hippocampus using cerebral blood volume (CBV)-based functional magnetic resonance imaging (fMRI). Basal CBV0 levels were similar between mEH E404D and control mice in both brain areas. But vascular reactivity and vasodilation in response to the vasodilatory drug acetazolamide were reduced in mEH E404D forebrain compared to WT controls by factor 3 and 2.6, respectively. These results demonstrate a critical role for mEH E404D in vasodynamics and suggest that deregulation of endogenous signaling pathways is the undesirable gain of function associated with the E404D variant.

  7. Effect of N-acetylcysteine in COPD patients with different microsomal epoxide hydrolase genotypes.

    PubMed

    Zhang, Jian-Qing; Zhang, Jia-Qiang; Liu, Hua; Zhao, Zhi-Huan; Fang, Li-Zhou; Liu, Ling; Fu, Wei-Ping; Shu, Jing-Kui; Feng, Jia-Gang; Dai, Lu-Ming

    2015-01-01

    The role of the antioxidant N-acetylcysteine (NAC) in the treatment of chronic obstructive pulmonary disease (COPD) has not been clarified as yet. In early studies, we found that the proportion of smokers with COPD having extremely slow/slow microsomal epoxide hydrolase (EPHX1) enzyme activity is significantly higher than that in healthy smokers. The purpose of this study was to evaluate whether different EPHX1 enzyme activity is related to differential therapeutic effects of treatment with NAC in COPD. A total of 219 patients with COPD were randomly allocated to an extremely slow/slow EPHX1 enzyme activity group (n=157) or a fast/normal EPHX1 enzyme activity group (n=62) according to their EPHX1 enzyme activity. Both groups were treated with NAC 600 mg twice daily for one year. The main study parameters, including forced expiratory volume in one second (FEV1), St George's Respiratory Questionnaire (SGRQ), and yearly exacerbation rate, were measured at baseline and at 6-month intervals for one year. Both FEV1 and SGRQ symptom scores were improved after treatment with NAC in the slow activity group when compared with the fast activity group. Further, changes in FEV1 and SGRQ symptom score in patients with mild-to-moderate COPD were more significant than those in patients with severe-to-very severe COPD. The yearly exacerbation rates were reduced in both groups, but the reduction in the slow activity group was significantly lower than in the fast activity group. NAC treatment in COPD patients with extremely slow/slow EPHX1 enzyme activity improves FEV1 and the SGRQ symptom score, especially in those with mild-to-moderate COPD, and polymorphism in the EPHX1 gene may have a significant role in differential responses to treatment with NAC in patients with COPD.

  8. Anti-Ulcer Efficacy of Soluble Epoxide Hydrolase Inhibitor TPPU on Diclofenac-Induced Intestinal Ulcers

    PubMed Central

    Goswami, Sumanta Kumar; Wan, Debin; Yang, Jun; Trindade da Silva, Carlos A.; Morisseau, Christophe; Kodani, Sean D.; Yang, Guang-Yu; Inceoglu, Bora

    2016-01-01

    Proton pump inhibitors such as omeprazole (OME) reduce the severity of gastrointestinal (GI) ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs) but can also increase the chance of dysbiosis. The aim of this study was to test the hypothesis that preventive use of a soluble epoxide hydrolase inhibitor (sEHI) such as TPPU can decrease NSAID-induced ulcers by increasing anti-inflammatory epoxyeicosatrienoic acids (EETs). Dose- [10, 30, and 100 mg/kg, by mouth (PO)] and time-dependent (6 and 18 hours) ulcerative effects of diclofenac sodium (DCF, an NSAID) were studied in the small intestine of Swiss Webster mice. Dose-dependent effects of TPPU (0.001–0.1 mg/kg per day for 7 days, in drinking water) were evaluated in DCF-induced intestinal toxicity and compared with OME (20 mg/kg, PO). In addition, the effect of treatment was studied on levels of Hb in blood, EETs in plasma, inflammatory markers such as myeloperoxidase (MPO) in intestinal tissue homogenates, and tissue necrosis factor-α (TNF-α) in serum. DCF dose dependently induced ulcers that were associated with both a significant (P < 0.05) loss of Hb and an increase in the level of MPO and TNF-α, with severity of ulceration highest at 18 hours. Pretreatment with TPPU dose dependently prevented ulcer formation by DCF, increased the levels of epoxy fatty acids, including EETs, and TPPU’s efficacy was comparable to OME. TPPU significantly (P < 0.05) reversed the effect of DCF on the level of Hb, MPO, and TNF-α. Thus sEHI might be useful in the management of NSAID-induced ulcers. PMID:26989141

  9. EET-dependent potentiation of pulmonary arterial pressure: sex-different regulation of soluble epoxide hydrolase

    PubMed Central

    Kandhi, Sharath; Qin, Jun; Froogh, Ghezal; Jiang, Houli; Luo, Meng; Wolin, Michael S.; Huang, An

    2015-01-01

    We tested the hypothesis that suppression of epoxyeicosatrienoic acid (EET) metabolism via genetic knockout of the gene for soluble epoxide hydrolase (sEH-KO), or female-specific downregulation of sEH expression, plays a role in the potentiation of pulmonary hypertension. We used male (M) and female (F) wild-type (WT) and sEH-KO mice; the latter have high pulmonary EETs. Right ventricular systolic pressure (RVSP) and mean arterial blood pressure (MABP) in control and in response to in vivo administration of U46619 (thromboxane analog), 14,15-EET, and 14,15-EEZE [14,15-epoxyeicosa-5(z)-enoic acid; antagonist of EETs] were recorded. Basal RVSP was comparable among all groups of mice, whereas MABP was significantly lower in F-WT than M-WT mice and further reduced predominantly in F-KO compared with M-KO mice. U46619 dose dependently increased RVSP and MABP in all groups of mice. The increase in RVSP was significantly greater and coincided with smaller increases in MABP in M-KO and F-WT mice compared with M-WT mice. In F-KO mice, the elevation of RVSP by U46619 was even higher than in M-KO and F-WT mice, associated with the least increase in MABP. 14,15-EEZE prevented the augmentation of U46619-induced elevation of RVSP in sEH-KO mice, whereas 14,15-EET-induced pulmonary vasoconstriction was comparable in all groups of mice. sEH expression in the lungs was reduced, paralleled with higher levels of EETs in F-WT compared with M-WT mice. In summary, EETs initiate pulmonary vasoconstriction but act as vasodilators systemically. High pulmonary EETs, as a function of downregulation or deletion of sEH, potentiate U46619-induced increases in RVSP in a female-susceptible manner. PMID:26498250

  10. Use of a soluble epoxide hydrolase inhibitor as adjunctive analgesic in a laminitic horse

    PubMed Central

    Guedes, Alonso G. P.; Morisseau, Christophe; Sole, Albert; Soares, Joao H. N.; Ulu, Arzu; Dong, Hua; Hammock, Bruce D.

    2014-01-01

    A 4-year old, 500 kg Thoroughbred female horse diagnosed with bilateral forelimb laminitis and cellulitis on the left forelimb became severely painful and refractory to non-steroidal anti-inflammatory therapy (flunixin meglumine on days 1, 2, 3 and 4; and phenylbutazone on days 5, 6 and 7) alone or in combination with gabapentin (days 6 and 7). Pain scores assessed independently by three individuals with a visual analog scale (VAS; 0= no pain and 10=worst possible pain) were 8.5 on day 6, and it increased to 9.5 on day 7. Non-invasive blood pressure monitoring revealed severe hypertension. As euthanasia was being considered for humane reasons as well as technical and financial constraints, a decision was made to add an experimental new drug, trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB), which is an inhibitor of soluble epoxide hydrolase (sEH), to the treatment protocol. Dose and frequency of administration were selected to produce plasma concentrations within the range of 2.5 μM and 30 nM based on the drug potency against equine sEH. Pain scores decreased sharply and remarkably following t-TUCB administration and blood pressure progressively decreased to physiologic normal values. Plasma concentrations of t-TUCB, measured daily, were within the expected range, whereas phenylbutazone and gabapentin plasma levels were below the suggested efficacious concentrations. No adverse effects were detected on clinical and laboratory examinations during and after t-TUCB administration. The mare did not get any episode of laminitis in the three months following the treatment. PMID:23463912

  11. Sexually dimorphic phenotype of arteriolar responsiveness to shear stress in soluble epoxide hydrolase-knockout mice

    PubMed Central

    Qin, Jun; Kandhi, Sharath; Froogh, Ghezal; Jiang, Houli; Luo, Meng; Sun, Dong

    2015-01-01

    We hypothesized that potentiating the bioavailability of endothelial epoxyeicosatrienoic acids (EETs) via deletion of the gene for soluble epoxide hydrolase (sEH), or downregulation of sEH expression, enhances flow/shear stress-induced dilator responses (FID) of arterioles. With the use of male (M) and female (F) wild-type (WT) and sEH-knockout (KO) mice, isolated gracilis muscle arterioles were cannulated and pressurized at 80 mmHg. Basal tone and increases in diameter of arterioles as a function of perfusate flow (5, 10, 15, 20, and 25 μl/min) were recorded. The magnitude of FID was significantly smaller and associated with a greater arteriolar tone in M-WT than F-WT mice, revealing a sex difference in FID. This sex difference was abolished by deletion of the sEH gene, as evidenced by an enhanced FID in M-KO mice to a level comparable with those observed in F-KO and F-WT mice. These three groups of mice coincidentally exhibited an increased endothelial sensitivity to shear stress (smaller WSS50) and were hypotensive. Endothelial EETs participated in the mediation of enhanced FID in M-KO, F-KO, and F-WT mice, without effects on FID of M-WT mice. Protein expression of sEH was downregulated by approximately fourfold in vessels of F-WT compared with M-WT mice, paralleled with greater vascular EET levels that were statistically comparable with those observed in both male and female sEH-KO mice. In conclusion, sex-different regulation of sEH accounts for sex differences in flow-mediated dilation of microvessels in gonadally intact mice. PMID:26453332

  12. Impact of epoxide hydrolase 1 polymorphisms on lung cancer susceptibility in Asian populations.

    PubMed

    Yu, Wei; Lin, Zhi; Qu, Baolin

    2015-03-01

    Inconsistent association of microsomal epoxide hydrolase (mEH) polymorphisms (Tyr113His, His139Arg) and lung cancer susceptibility have been reported in earlier studies. This study was undertaken to assess if mEH Tyr113His and His139Arg represent risk factors for lung cancer in Asian population. We exhaustively searched multiple databases to identify all eligible studies. Odds ratios were calculated to estimate the strength of genetic associations. This meta-analysis finally combined 2,522 subjects for Tyr113His and 2,725 subjects for His139Arg. In the analysis of Tyr113His, the His/His genotype carriers were found to have 29 % higher risk of lung cancer compared to the Tyr/Tyr carriers (His/His vs. Tyr/Tyr, odds ratio, 1.29, 95 % confidence interval, 1.06-1.58). A significantly increased risk was also seen in His/His versus His/Tyr + Tyr/Tyr (odds ratio, 1.29, 95 % confidence interval, 1.07-1.55). Likewise, His139Arg demonstrated a significant association with lung cancer (Arg/His vs. His/His, odds ratio, 1.2 6, 95 % confidence interval, 1.06-1.49; odds ratio, 1.24, 95 % confidence interval, 1.05-1.46). Stratified analysis by ethnicity showed both of the polymorphisms were associated with lung cancer in Chinese populations. These results suggest that the genetic associations exist between mEH polymorphisms and lung cancer susceptibility in Asian populations.

  13. Altered soluble epoxide hydrolase-derived oxylipins in patients with seasonal major depression: An exploratory study.

    PubMed

    Hennebelle, Marie; Otoki, Yurika; Yang, Jun; Hammock, Bruce D; Levitt, Anthony J; Taha, Ameer Y; Swardfager, Walter

    2017-06-01

    Many cytochrome p450-derived lipids promote resolution of inflammation, in contrast to their soluble epoxide hydrolase(sEH)-derived oxylipin breakdown products. Here we compare plasma oxylipins and precursor fatty acids between seasons in participants with major depressive disorder with seasonal pattern (MDD-s). Euthymic participants with a history of MDD-s recruited in summer-fall were followed-up in winter. At both visits, a structured clinical interview (DSM-5 criteria) and the Beck Depression Inventory II (BDI-II) were administered. Unesterified and total oxylipin pools were assayed by liquid chromatography tandem mass-spectrometry (LC-MS/MS). Precursor fatty acids were measured by gas chromatography. In nine unmedicated participants euthymic at baseline who met depression criteria in winter, BDI-II scores increased from 4.9±4.4 to 19.9±7.7. Four sEH-derived oxylipins increased in winter compared to summer-fall with moderate to large effect sizes. An auto-oxidation product (unesterified epoxyketooctadecadienoic acid) and lipoxygenase-derived 13-hydroxyoctadecadienoic acid also increased in winter. The cytochrome p450-derived 20-COOH-leukotriene B4 (unesterified) and total 14(15)-epoxyeicosatetraenoic acid, and the sEH-derived 14,15-dihydroxyeicostrienoic acid (unesterified), decreased in winter. We conclude that winter depression was associated with changes in cytochrome p450- and sEH-derived oxylipins, suggesting that seasonal shifts in omega-6 and omega-3 fatty acid metabolism mediated by sEH may underlie inflammatory states in symptomatic MDD-s. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  14. In vitro and in vivo metabolism of N-adamantyl substituted urea-based soluble epoxide hydrolase inhibitors.

    PubMed

    Liu, Jun-Yan; Tsai, Hsing-Ju; Morisseau, Christophe; Lango, Jozsef; Hwang, Sung Hee; Watanabe, Takaho; Kim, In-Hae; Hammock, Bruce D

    2015-12-15

    N,N'-disubstituted urea-based soluble epoxide hydrolase (sEH) inhibitors are promising therapeutics for hypertension, inflammation, and pain in multiple animal models. The drug absorption and pharmacological efficacy of these inhibitors have been reported extensively. However, the drug metabolism of these inhibitors is not well described. Here we reported the metabolic profile and associated biochemical studies of an N-adamantyl urea-based sEH inhibitor 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)pentyl)urea (AEPU) in vitro and in vivo. The metabolites of AEPU were identified by interpretation of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or NMR. In vitro, AEPU had three major positions for phase I metabolism including oxidations on the adamantyl moiety, urea nitrogen atoms, and cleavage of the polyethylene glycol chain. In a rodent model, the metabolites from the hydroxylation on the adamantyl group and nitrogen atom were existed in blood while the metabolites from cleavage of polyethylene glycol chain were not found in urine. The major metabolite found in rodent urine was 3-(3-adamantyl-ureido)-propanoic acid, a presumably from cleavage and oxidation of the polyethylene glycol moiety. All the metabolites found were active but less potent than AEPU at inhibiting human sEH. Furthermore, cytochrome P450 (CYP) 3A4 was found to be a major enzyme mediating AEPU metabolism. In conclusion, the metabolism of AEPU resulted from oxidation by CYP could be shared with other N-adamantyl-urea-based compounds. These findings suggest possible therapeutic roles for AEPU and new strategies for drug design in this series of possible drugs.

  15. Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE2 induced pain model

    PubMed Central

    Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D.; da Silva, Carlos Antonio Trindade; Morisseau, Christophe; Hammock, Bruce D.

    2015-01-01

    Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE2 was monitored. While OME treatment by itself exhibited variable effects on PGE2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. PMID:26522832

  16. Structure-based optimization of the piperazino-containing 1,3-disubstituted ureas affording sub-nanomolar inhibitors of soluble epoxide hydrolase

    PubMed Central

    Huang, Shao-Xu; Cao, Bin; Morisseau, Christophe; Jin, Yi; Hammock, Bruce D.; Long, Ya-Qiu

    2014-01-01

    The inhibition of the soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hypertension, inflammation and other cardiovascular disorders. Piperazino functionality as the tertiary pharmacophore remarkably improved the drug-like profile of the 1,3-disubstituted urea sEH inhibitors. However, the potency was more dependent on the overall best balance of the hydrophilicity and lipophilicity. Based on the sEH-inhibitor complex structure, further structural optimization on the piperazino-containing 1,3-disubstituted urea scaffold was conducted for an improved potency. The 1-adamantylacetamide and para-phenylcarbonyl group were identified to be an optimal primary pharmacophore and secondary pharmacophore motif, respectively, generating sub-nanomolar sEH inhibitors with favorable water solubility. PMID:25225606

  17. Structure-based optimization of the piperazino-containing 1,3-disubstituted ureas affording sub-nanomolar inhibitors of soluble epoxide hydrolase.

    PubMed

    Huang, Shao-Xu; Cao, Bin; Morisseau, Christophe; Jin, Yi; Hammock, Bruce D; Long, Ya-Qiu

    2012-03-01

    The inhibition of the soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hypertension, inflammation and other cardiovascular disorders. Piperazino functionality as the tertiary pharmacophore remarkably improved the drug-like profile of the 1,3-disubstituted urea sEH inhibitors. However, the potency was more dependent on the overall best balance of the hydrophilicity and lipophilicity. Based on the sEH-inhibitor complex structure, further structural optimization on the piperazino-containing 1,3-disubstituted urea scaffold was conducted for an improved potency. The 1-adamantylacetamide and para-phenylcarbonyl group were identified to be an optimal primary pharmacophore and secondary pharmacophore motif, respectively, generating sub-nanomolar sEH inhibitors with favorable water solubility.

  18. 1,3-Disubstituted Ureas Functionalized with Ether Groups are Potent Inhibitors of the Soluble Epoxide Hydrolase with Improved Pharmacokinetic Properties

    PubMed Central

    Kim, In-Hae; Tsai, Hsing-Ju; Nishi, Kosuke; Kasagami, Takeo; Morisseau, Christophe; Hammock, Bruce D.

    2008-01-01

    Soluble epoxide hydrolase (sEH) is a therapeutic target for treating hypertension and inflammation. 1,3-Disubstituted ureas functionalized with an ether group are potent sEH inhibitors. However, their relatively low metabolic stability leads to poor pharmacokinetic properties. To improve their bioavailability, we investigated the effect of incorporating various polar groups on the ether function on the inhibition potencies, physical properties, in vitro metabolic stability, and pharmacokinetic properties. The structure-activity relationship (SAR) studies showed that a hydrophobic linker between the urea group and the ether function is necessary to keep their potency. In addition, urea-ether inhibitors having a polar group such as diethylene glycol or morpholine significantly improved their physical properties and metabolic stability without any loss of inhibitory potency. Furthermore, improved pharmacokinetic properties in murine and canine models were obtained with the resulting inhibitors. These findings will facilitate the usage of sEH inhibitors in animal models of hypertension and inflammation. PMID:17894481

  19. The 2014 Bernard B. Brodie Award Lecture—Epoxide Hydrolases: Drug Metabolism to Therapeutics for Chronic Pain

    PubMed Central

    Kodani, Sean D.

    2015-01-01

    Dr. Bernard Brodie’s legacy is built on fundamental discoveries in pharmacology and drug metabolism that were then translated to the clinic to improve patient care. Similarly, the development of a novel class of therapeutics termed the soluble epoxide hydrolase (sEH) inhibitors was originally spurred by fundamental research exploring the biochemistry and physiology of the sEH. Here, we present an overview of the history and current state of research on epoxide hydrolases, specifically focusing on sEHs. In doing so, we start with the translational project studying the metabolism of the insect juvenile hormone mimic R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene], which led to the identification of the mammalian sEH. Further investigation of this enzyme and its substrates, including the epoxyeicosatrienoic acids, led to insight into mechanisms of inflammation, chronic and neuropathic pain, angiogenesis, and other physiologic processes. This basic knowledge in turn led to the development of potent inhibitors of the sEH that are promising therapeutics for pain, hypertension, chronic obstructive pulmonary disorder, arthritis, and other disorders. PMID:25762541

  20. Exposure to ethylene oxide in hospitals: biological monitoring and influence of glutathione S-transferase and epoxide hydrolase polymorphisms.

    PubMed

    Haufroid, Vincent; Merz, Brigitte; Hofmann, Annette; Tschopp, Alois; Lison, Dominique; Hotz, Philippe

    2007-04-01

    Ethylene oxide is considered as a human carcinogen. A biomarker of exposure would be a useful instrument to assess the risk in occupationally exposed workers. This cross-sectional study aimed at examining (a) whether the urinary excretion of a metabolite of ethylene oxide, 2-hydroxyethyl mercapturic acid (HEMA), could be used for monitoring occupational exposure and (b) whether glutathione S-transferase (GST) and epoxide hydrolase genotypes influenced biological monitoring. Exposure to ethylene oxide was measured by personal sampling in 80 hospital workers (95% of those eligible). HEMA concentrations were determined in three urine samples (baseline, end of shift, and next morning) by liquid chromatography with tandem mass spectrometry. GSTs (GSTT1, GSTM1, and GSTP1) and epoxide hydrolase (EPHX1) were also genotyped. The influence of exposure, genotypes, and several other factors was examined in multiple regression analyses. Exposure was always <1 parts per million. On a group basis, exposure and a non-null GSTT1 genotype increased the HEMA concentrations in the urine sample collected at the end of the shift and these factors remained statistically significant after considering possible confounding or modifying factors.

  1. Soluble Epoxide Hydrolase Inhibitors Reduce the Development of Atherosclerosis in Apolipoprotein E-Knockout Mouse Model

    PubMed Central

    Ulu, Arzu; Davis, Benjamin B.; Tsai, Hsing-Ju; Kim, In-Hae; Morisseau, Christophe; Inceoglu, Bora; Fiehn, Oliver; Hammock, Bruce D.; Weiss, Robert H.

    2008-01-01

    In order to determine whether sEH inhibitors influence atherosclerotic lesion formation, we utilized an established murine model of accelerated atherogenesis, ApoE knockout (−/−) mice. The sEH inhibitor, 1-adamantan-3-(5-(2-(2-ethylethoxy)ethoxy)pentyl)urea (AEPU) was delivered in drinking water. All animals were fed an atherogenic diet while simultaneously infused with angiotensin II by osmotic minipump to induce atherosclerosis. In AEPU-treated animals, there was a 53% reduction in atherosclerotic lesions in the descending aortae as compared to control aortae. AEPU and its major metabolites were detected in the plasma of animals which received it. As expected from the inhibition of sEH, a significant increase in linoleic and arachidonic acid epoxides, as well as an increase in individual 11,12-EET/DHET and 14,15-EET/DHET ratios, were observed. The reduction in atherosclerotic lesion area was inversely correlated with 11,12- and 14,15- EET/DHET ratios, suggesting that the reduction corresponds to the inhibition of sEH. Our data suggest that orally-available sEH inhibitors may be useful in the treatment of patients with atherosclerotic cardiovascular disease. PMID:18791465

  2. Nanobody based immunoassay for human soluble epoxide hydrolase detection using polyHRP for signal enhancement—the rediscovery of polyHRP

    USDA-ARS?s Scientific Manuscript database

    Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain and multiple cardiovascular related diseases. A variable domain of a heavy chain only antibody (termed sdAb, nanobody or VHH) possesses advantages of small size, high ...

  3. ADMINISTRATION OF A SUBSTITUTED ADAMANTLY-UREA INHIBITOR OF THE SOLUBLE EPOXIDE HYDROLASE PROTECTS THE KIDNEY FROM DAMAGE IN HYPERTENSIVE GOTO-KAKIZAKI RATS

    USDA-ARS?s Scientific Manuscript database

    Hypertension and type II diabetes are co-morbid diseases that lead to the development of nephropathy. Soluble epoxide hydrolase (sEH) inhibitors are reported to provide protection from renal injury. We hypothesized that the sEH inhibitor 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) protects ...

  4. Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor

    PubMed Central

    Stsiapanava, Alena; Olsson, Ulrika; Wan, Min; Kleinschmidt, Thea; Rutishauser, Dorothea; Zubarev, Roman A.; Samuelsson, Bengt; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z.

    2014-01-01

    Leukotriene (LT) A4 hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB4. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA4 hydrolase. Here, we determined the crystal structure of LTA4 hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA4 hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB4 synthesis in human neutrophils (IC50 of ∼0.5 μM) and conversion of LTA4 into LTB4 by purified LTA4H with a Ki of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA4 hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA4, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA4 into LTB4, although sparing the enzyme’s anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA4 hydrolase inhibitor that holds promise for improved anti-inflammatory properties. PMID:24591641

  5. Discovery of potent inhibitors of soluble epoxide hydrolase by combinatorial library design and structure-based virtual screening.

    PubMed

    Xing, Li; McDonald, Joseph J; Kolodziej, Steve A; Kurumbail, Ravi G; Williams, Jennifer M; Warren, Chad J; O'Neal, Janet M; Skepner, Jill E; Roberds, Steven L

    2011-03-10

    Structure-based virtual screening was applied to design combinatorial libraries to discover novel and potent soluble epoxide hydrolase (sEH) inhibitors. X-ray crystal structures revealed unique interactions for a benzoxazole template in addition to the conserved hydrogen bonds with the catalytic machinery of sEH. By exploitation of the favorable binding elements, two iterations of library design based on amide coupling were employed, guided principally by the docking results of the enumerated virtual products. Biological screening of the libraries demonstrated as high as 90% hit rate, of which over two dozen compounds were single digit nanomolar sEH inhibitors by IC(50) determination. In total the library design and synthesis produced more than 300 submicromolar sEH inhibitors. In cellular systems consistent activities were demonstrated with biochemical measurements. The SAR understanding of the benzoxazole template provides valuable insights into discovery of novel sEH inhibitors as therapeutic agents.

  6. Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

    PubMed Central

    Temml, Veronika; Garscha, Ulrike; Romp, Erik; Schubert, Gregor; Gerstmeier, Jana; Kutil, Zsofia; Matuszczak, Barbara; Waltenberger, Birgit; Stuppner, Hermann; Werz, Oliver; Schuster, Daniela

    2017-01-01

    Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N’-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay. PMID:28218273

  7. Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

    NASA Astrophysics Data System (ADS)

    Temml, Veronika; Garscha, Ulrike; Romp, Erik; Schubert, Gregor; Gerstmeier, Jana; Kutil, Zsofia; Matuszczak, Barbara; Waltenberger, Birgit; Stuppner, Hermann; Werz, Oliver; Schuster, Daniela

    2017-02-01

    Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N’-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay.

  8. Administration of a substituted adamantyl urea inhibitor of soluble epoxide hydrolase protects the kidney from damage in hypertensive Goto-Kakizaki rats.

    PubMed

    Olearczyk, Jeffrey J; Quigley, Jeffrey E; Mitchell, Bradford C; Yamamoto, Tatsuo; Kim, In-Hae; Newman, John W; Luria, Ayala; Hammock, Bruce D; Imig, John D

    2009-01-01

    Hypertension and Type 2 diabetes are co-morbid diseases that lead to the development of nephropathy. sEH (soluble epoxide hydrolase) inhibitors are reported to provide protection from renal injury. We hypothesized that the sEH inhibitor AUDA [12-(3-adamantan-1-yl-ureido)-dodecanoic acid] protects the kidney from the development of nephropathy associated with hypertension and Type 2 diabetes. Hypertension was induced in spontaneously diabetic GK (Goto-Kakizaki) rats using AngII (angiotensin II) and a high-salt diet. Hypertensive GK rats were treated for 2 weeks with either AUDA or its vehicle added to drinking water. MAP (mean arterial pressure) increased from 118+/-2 mmHg to 182+/-20 and 187+/-6 mmHg for vehicle and AUDA-treated hypertensive GK rats respectively. AUDA treatment did not alter blood glucose. Hypertension in GK rats resulted in a 17-fold increase in urinary albumin excretion, which was decreased with AUDA treatment. Renal histological evaluation determined that AUDA treatment decreased glomerular and tubular damage. In addition, AUDA treatment attenuated macrophage infiltration and inhibited urinary excretion of MCP-1 (monocyte chemoattractant protein-1) and kidney cortex MCP-1 gene expression. Taken together, these results provide evidence that sEH inhibition with AUDA attenuates the progression of renal damage associated with hypertension and Type 2 diabetes.

  9. Anti-inflammatory effects of ω-3 polyunsaturated fatty acids and soluble epoxide hydrolase inhibitors in angiotensin-II-dependent hypertension.

    PubMed

    Ulu, Arzu; Harris, Todd R; Morisseau, Christophe; Miyabe, Christina; Inoue, Hiromi; Schuster, Gertrud; Dong, Hua; Iosif, Ana-Maria; Liu, Jun-Yan; Weiss, Robert H; Chiamvimonvat, Nipavan; Imig, John D; Hammock, Bruce D

    2013-09-01

    The mechanisms underlying the anti-inflammatory and antihypertensive effects of long-chain ω-3 polyunsaturated fatty acids (ω-3 PUFAs) are still unclear. The epoxides of an ω-6 fatty acid, arachidonic acid epoxyeicosatrienoic acids also exhibit antihypertensive and anti-inflammatory effects. Thus, we hypothesized that the major ω-3 PUFAs, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may lower the blood pressure and attenuate renal markers of inflammation through their epoxide metabolites. Here, we supplemented mice with an ω-3 rich diet for 3 weeks in a murine model of angiotensin-II-dependent hypertension. Also, because EPA and DHA epoxides are metabolized by soluble epoxide hydrolase (sEH), we tested the combination of an sEH inhibitor and the ω-3 rich diet. Our results show that ω-3 rich diet in combination with the sEH inhibitor lowered Ang-II, increased the blood pressure, further increased the renal levels of EPA and DHA epoxides, reduced renal markers of inflammation (ie, prostaglandins and MCP-1), downregulated an epithelial sodium channel, and upregulated angiotensin-converting enzyme-2 message and significantly modulated cyclooxygenase and lipoxygenase metabolic pathways. Overall, our findings suggest that epoxides of the ω-3 PUFAs contribute to lowering systolic blood pressure and attenuating inflammation in part by reduced prostaglandins and MCP-1 and by upregulation of angiotensin-converting enzyme-2 in angiotensin-II-dependent hypertension.

  10. Anti-inflammatory Effects of Omega-3 Polyunsaturated Fatty Acids and Soluble Epoxide Hydrolase Inhibitors in Angiotensin-II Dependent Hypertension

    PubMed Central

    Ulu, Arzu; Harris, Todd R; Morisseau, Christophe; Miyabe, Christina; Inoue, Hiromi; Schuster, Gertrud; Dong, Hua; Iosif, Ana-Maria; Liu, Jun-Yan; Weiss, Robert H; Chiamvimonvat, Nipavan; Imig, John D; Hammock, Bruce D

    2013-01-01

    The mechanisms underlying the anti-inflammatory and anti-hypertensive effects of long chain ω-3 polyunsaturated fatty acids (PUFAs) are still unclear. The epoxides of an ω-6 fatty acid, arachidonic acid (epoxyeicosatrienoic acids; EETs) also exhibit anti-hypertensive and anti-inflammatory effects. Thus, we hypothesized that the major ω-3 PUFAs including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may lower blood pressure and attenuate renal markers of inflammation through their epoxide metabolites. Here, we supplemented mice with an ω-3 rich diet for three weeks in a murine model of angiotensin-II dependent hypertension. Also, since EPA and DHA epoxides are metabolized by soluble epoxide hydrolase (sEH), we tested the combination of a sEH inhibitor and the ω-3 rich diet. Our results show that ω-3 rich diet in combination with the sEH inhibitor lowered Ang-II increased blood pressure, further increased renal levels of EPA and DHA epoxides, reduced renal markers of inflammation (i.e. prostaglandins and MCP-1), down-regulated an epithelial sodium channel and up-regulated Angiotensin converting enzyme-2 message (ACE-2) and significantly modulated cyclooxygenase and lipoxygenase metabolic pathways. Overall, our findings suggest that epoxides of the ω-3 PUFAs contribute to lowering SBP and attenuating inflammation in part by reduced prostaglandins and MCP-1 and by up-regulation of ACE-2 in angiotensin-II dependent hypertension. PMID:23676336

  11. Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor▿ †

    PubMed Central

    Bahl, Christopher D.; Morisseau, Christophe; Bomberger, Jennifer M.; Stanton, Bruce A.; Hammock, Bruce D.; O'Toole, George A.; Madden, Dean R.

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other α/β hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-Å resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of α/β hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate. PMID:20118260

  12. Crystal structure of the cystic fibrosis transmembrane conductance regulator inhibitory factor Cif reveals novel active-site features of an epoxide hydrolase virulence factor.

    PubMed

    Bahl, Christopher D; Morisseau, Christophe; Bomberger, Jennifer M; Stanton, Bruce A; Hammock, Bruce D; O'Toole, George A; Madden, Dean R

    2010-04-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other alpha/beta hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-A resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of alpha/beta hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

  13. Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics.

    PubMed

    Archelas, Alain; Zhao, Wei; Faure, Bruno; Iacazio, Gilles; Kotik, Michael

    2016-02-01

    A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the α/β-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of ∼200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K(M) values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k2/K(S)) and the alkyl-enzyme hydrolysis rate k3 between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate.

  14. Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

    SciTech Connect

    Bahl, C.; Morisseau, C; Bomberger, J; Stanton, B; Hammock, B; O' Toole, G; Madden, D

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other {alpha}/{beta} hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-{angstrom} resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of {alpha}/{beta} hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

  15. Development of Monoclonal Antibodies to Human Microsomal Epoxide Hydrolase and Analysis of “Preneoplastic Antigen”-Like Molecules

    PubMed Central

    Duan, Hongying; Yoshimura, Kazunori; Kobayashi, Nobuharu; Sugiyama, Kazuo; Sawada, Jun-ichi; Saito, Yoshiro; Morisseau, Christophe; Hammock, Bruce D.; Akatsuka, Toshitaka

    2012-01-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one, anti-C-terminal; one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. PMID:22310175

  16. Transcription of the human microsomal epoxide hydrolase gene (EPHX1) is regulated by an HNF-4α/CAR/RXR/PSF complex.

    PubMed

    Peng, Hui; Zhu, Qin-Shi; Zhong, Shuping; Levy, Daniel

    2013-10-01

    Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes where they are involved in cholesterol excretion and metabolism, lipid digestion and regulating numerous signaling pathways. Previous studies have demonstrated the critical role of GATA-4 and a C/EBPα-NF/Y complex in the regulation of the mEH gene (EPHX1). In this study we show that HNF-4α and CAR/RXR also bind to the proximal promoter region and regulate EPHX1 expression. Bile acids, which inhibit the expression of HNF-4α also decrease the expression of EPHX1. Studies also established that the binding of HNF-4α was essential for the activation of EPHX1 activity by CAR suggesting the formation of a complex between these adjacent factors. The nature of this regulatory complex was further explored using a biotinylated oligonucleotide of this region in conjunction with BioMag beads and mass spectrometric analysis which demonstrated the presence of an additional inhibitory factor (PSF), confirmed by co-immunoprecipitation and ChIP analyses, which interacted with DNA-bound CAR/RXR/HNF-4α forming a 4-component regulatory complex. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Potent Natural Soluble Epoxide Hydrolase Inhibitors from Pentadiplandra brazzeana Baillon: Synthesis, Quantification, and Measurement of Biological Activities In Vitro and In Vivo

    PubMed Central

    Kitamura, Seiya; Morisseau, Christophe; Inceoglu, Bora; Kamita, Shizuo G.; De Nicola, Gina R.; Nyegue, Maximilienne; Hammock, Bruce D.

    2015-01-01

    We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most abundant (42.3 μg/g dry root weight). All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana. PMID:25659109

  18. Computational study of phosphatase activity in soluble epoxide hydrolase: high efficiency through a water bridge mediated proton shuttle.

    PubMed

    De Vivo, Marco; Ensing, Bernd; Klein, Michael L

    2005-08-17

    Recently, a new branch of fatty acid metabolism has been opened by the novel phosphatase activity found in the N-terminal domain of the, hence bifunctional, soluble epoxide hydrolase (sEH). Importantly, this finding has also provided a new site for drug targeting in sEH's activity regulation. Classical MD and hybrid Car-Parrinello QM/MM calculations have been performed to investigate the reaction mechanism of the phosphoenzyme intermediate formation in the first step of the catalysis. The results support a concerted multi-event reaction mechanism: (1) a dissociative in-line nucleophilic substitution for the phosphoryl transfer reaction; (2) a double proton transfer involved in the formation of a good leaving group in the transition state. The presence of a water bridge in the substrate/enzyme complex allowed an efficient proton shuttle, showing its key role in speeding up the catalysis. The calculated free energy of the favored catalytic pathway is approximately 19 kcal/mol, in excellent agreement with experimental data.

  19. Role of soluble epoxide hydrolase in exacerbation of stroke by streptozotocin-induced type 1 diabetes mellitus.

    PubMed

    Jouihan, Sari A; Zuloaga, Kristen L; Zhang, Wenri; Shangraw, Robert E; Krasnow, Stephanie M; Marks, Daniel L; Alkayed, Nabil J

    2013-10-01

    Hyperglycemia worsens stroke, yet rigorous glycemic control does not improve neurologic outcome. An alternative is to target downstream molecular mediator(s) triggered by hyperglycemia but independent of prevailing glycemia. Soluble epoxide hydrolase (sEH) is a potential mediator of injury via its metabolism of neuroprotective epoxyeicosatrienoic acids (EETs). We tested whether hyperglycemia exacerbates cerebral injury by upregulating sEH and decreasing brain EET levels. Type 1 diabetes mellitus was modeled by streptozotocin (STZ; 50 mg/kg per day intraperitoneally, 5 days) in male mice. At 4 weeks, STZ-treated and control mice underwent 45-minute middle cerebral artery occlusion (MCAO) with or without sEH blockade by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 1 mg/kg intraperitoneally daily for 6 days before MCAO). The STZ-treated mice had increased sEH mRNA expression in cerebral vessels and decreased EET concentrations in brain. There was no difference in cortical perfusion between groups. The STZ-treated mice sustained larger brain infarct than controls. Pretreatment with t-AUCB eliminated the difference in infarct size and EETs concentration between STZ-treated mice and controls, without altering glycemia. We conclude that type 1 diabetes mellitus upregulates sEH mRNA and decreases concentrations of neuroprotective EETs within the brain, leading to worse stroke outcome. The data indicate that sEH antagonism may be beneficial in the setting of hyperglycemic stroke.

  20. Enhancement of epoxide hydrolase production by (60) Co gamma and UV irradiation mutagenesis of Aspergillus niger ZJB-09103.

    PubMed

    Jin, Huo-Xi; OuYang, Xiao-Kun; Hu, Zhong-Ce

    2017-05-01

    An effective epoxide hydrolase (EH) production strain was mutagenized using (60) Co gamma and UV irradiation. Among positive mutant strains, the EH activity of C2-44 reached 33.7 U/g, which was 267% as much as that of the original Aspergillus niger ZJB-09103. Compared with the wild type, there were significant changes in morphology for C2-44, including the color of mycelia on the slants and the shape of conidial head. In addition, glucose and soybean cake were the optimal carbon and nitrogen source in terms of EH activity for the mutant C2-44 instead of soluble starch and peptone for the wild-type strain. The reaction time required to reach 99% enantiomeric excesses of (S)-epichlorohydrin from racemic substrate was shortened significantly by the mutant C2-44. This phenomenon was probably explained by the higher Vmax for hydrolysis of racemic epichlorohydrin by C2-44 compared with Aspergillus niger ZJB-09103. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  1. Single administration of soluble epoxide hydrolase inhibitor suppresses neuroinflammation and improves neuronal damage after cardiac arrest in mice.

    PubMed

    Taguchi, Noriko; Nakayama, Shin; Tanaka, Makoto

    2016-10-01

    Cardiac arrest (CA) causes ischemia-reperfusion injury in the whole body among victims. Especially in the brain, inflammation and neuronal cell death can lead to irreversible dysfunction. Our goal was to determine whether a single administration of soluble epoxide hydrolase inhibitor (AS2586144-CL) has a neuroprotective effect and decreases the inflammatory response after CA and cardiopulmonary resuscitation (CPR). Global cerebral ischemia was induced in male C57BL/6 mice with 8min of CA. Thirty minutes after recovery of spontaneous circulation, the mice were randomly assigned to three groups and administered AS2586144-CL: 1mg/kg (n=25), 10mg/kg (n=25), or 0mg/kg (vehicle, n=25). At 6 and 7 days after CA/CPR, behavioral tests were conducted and brains were removed for histological evaluation. Analysis of histological damage 7 days after CA/CPR revealed that 10mg/kg of AS2586144-CL protected neurons, and suppressed cytokine production and microglial migration into the hippocampus. Two hours after CA/CPR, 10mg/kg of AS2586144-CL suppressed serum tumor necrosis factor-α and hippocampal nuclear factor κB expression. Our data show that 10mg/kg of AS2586144-CL administered following CA/CPR suppresses inflammation and decreases neuronal damage.

  2. Discovering Drugs with DNA-Encoded Library Technology: From Concept to Clinic with an Inhibitor of Soluble Epoxide Hydrolase.

    PubMed

    Belyanskaya, Svetlana L; Ding, Yun; Callahan, James F; Lazaar, Aili L; Israel, David I

    2017-03-09

    DNA-encoded chemical library technology was developed with the vision of its becoming a transformational platform for drug discovery. The hope was that a new paradigm for the discovery of low-molecular-weight drugs would be enabled by combining the vast molecular diversity achievable with combinatorial chemistry, the information-encoding attributes of DNA, the power of molecular biology, and a streamlined selection-based discovery process. Here, we describe the discovery and early clinical development of GSK2256294, an inhibitor of soluble epoxide hydrolase (sEH, EPHX2), by using encoded-library technology (ELT). GSK2256294 is an orally bioavailable, potent and selective inhibitor of sEH that has a long half life and produced no serious adverse events in a first-time-in-human clinical study. To our knowledge, GSK2256294 is the first molecule discovered from this technology to enter human clinical testing and represents a realization of the vision that DNA-encoded chemical library technology can efficiently yield molecules with favorable properties that can be readily progressed into high-quality drugs.

  3. Fragment Screening of Soluble Epoxide Hydrolase for Lead Generation-Structure-Based Hit Evaluation and Chemistry Exploration.

    PubMed

    Xue, Yafeng; Olsson, Thomas; Johansson, Carina A; Öster, Linda; Beisel, Hans-Georg; Rohman, Mattias; Karis, David; Bäckström, Stefan

    2016-03-04

    Soluble epoxide hydrolase (sEH) is involved in the regulation of many biological processes by metabolizing the key bioactive lipid mediator, epoxyeicosatrienoic acids. For the development of sEH inhibitors with improved physicochemical properties, we performed both a fragment screening and a high-throughput screening aiming at an integrated hit evaluation and lead generation. Followed by a joint dose-response analysis to confirm the hits, the identified actives were then effectively triaged by a structure-based hit-classification approach to three prioritized series. Two distinct scaffolds were identified as tractable starting points for potential lead chemistry work. The oxoindoline series bind at the right-hand side of the active-site pocket with hydrogen bonds to the protein. The 2-phenylbenzimidazole-4-sulfonamide series bind at the central channel with significant induced fit, which has not been previously reported. On the basis of the encouraging initial results, we envision that a new lead series with improved properties could be generated if a vector is found that could merge the cyclohexyl functionality of the oxoindoline series with the trifluoromethyl moiety of the 2-phenylbenzimidazole-4-sulfonamide series.

  4. Investigation of the Association between Genetic Polymorphism of Microsomal Epoxide Hydrolase and Primary Brain Tumor Incidence

    PubMed Central

    Aydin, Ali; Pinarbasi, Hatice; Gurelik, Mustafa

    2013-01-01

    mEH is a critical biotransformation enzyme that catalyzes the conversion of xenobiotic epoxide substrates into more polar diol metabolites: it is also capable of inactivating a large number of structurally different molecules. Two polymorphisms affecting enzyme activity have been described in the exon 3 and 4 of the mEH gene. The hypothesis of this study is that inherent genetic susceptibility to a primary brain tumor is associated with mEH gene polymorphisms. The polymorphisms of the mEH gene were determined with PCR-RFLP techniques and 255 Turkish individuals. Our results indicate that the frequency of the mEH exon 4 polymorphism (in controls) is significantly higher than that of primary brain tumor patients (OR = 1.8, 95% CI = 1.0–3.4). This report, however, failed to demonstrate a significant association between mEH exon 3 polymorphism and primary brain tumor susceptibility in this population. Analysis of patients by both histological types of primary brain tumor and gene variants showed no association, although analysis of family history of cancer between cases and controls showed a statistically significant association (χ 2 = 7.0, P = 0.01). Our results marginally support the hypothesis that genetic susceptibility to brain tumors may be associated with mEPHX gene polymorphisms. PMID:24455257

  5. The juvenile hormone (JH) epoxide hydrolase gene in the honey bee (Apis mellifera) genome encodes a protein which has negligible participation in JH degradation.

    PubMed

    Mackert, Aline; Hartfelder, Klaus; Bitondi, Márcia Maria Gentile; Simões, Zilá Luz Paulino

    2010-09-01

    Epoxide hydrolases are multifunctional enzymes that are best known in insects for their role in juvenile hormone (JH) degradation. Enzymes involved in JH catabolism can play major roles during metamorphosis and reproduction, such as the JH epoxide hydrolase (JHEH), which degrades JH through hydration of the epoxide moiety to form JH diol, and JH esterase (JHE), which hydrolyzes the methyl ester to produce JH acid. In the honey bee, JH has been co-opted for additional functions, mainly in caste differentiation and in age-related behavioral development of workers, where the activity of both enzymes could be important for JH titer regulation. Similarity searches for jheh candidate genes in the honey bee genome revealed a single Amjheh gene. Sequence analysis, quantification of Amjheh transcript levels and Western blot assays using an AmJHEH-specific antibody generated during this study revealed that the AmJHEH found in the fat body shares features with the microsomal JHEHs from several insect species. Using a partition assay we demonstrated that AmJHEH has a negligible role in JH degradation, which, in the honey bee, is thus performed primarily by JHE. High AmJHEH levels in larvae and adults were related to the ingestion of high loads of lipids, suggesting that AmJHEH has a role in dietary lipid catabolism.

  6. Pseudomonas aeruginosa Cif defines a distinct class of α/β epoxide hydrolases utilizing a His/Tyr ring-opening pair

    PubMed Central

    Bahl, Christopher D.; Madden, Dean R.

    2012-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that secretes a multitude of virulence factors during the course of infection. Among these is Cif, an epoxide hydrolase (EH) that reduces the functional localization of the cystic fibrosis transmembrane conductance regulator in epithelial cells. In addition to being the first reported EH virulence factor, Cif possesses unique sequence deviations from canonical EH motifs. Foremost among these is the substitution of a histidine for the second epoxide ring-opening tyrosine in the active site. To test the functional equivalence of the Tyr and His side chains at this position, we have generated the mutant Cif-H177Y. Structural analysis confirms that both the WT His and mutant Tyr side chains can be accommodated without large-scale conformational changes. However, the Tyr mutation is functionally inactive. Based on a detailed analysis of the structure of the Tyr mutant, it appears that Cif's main-chain conformation imposes a functional requirement for a His at this position. Comparison with canonical EH structures reveals additional conformational differences, which are coupled to divergent sequence characteristics. When used to probe the genomes of other opportunistic pathogens, these sequence-structure criteria uncover candidate sequences that appear to form a distinct subfamily of Cif-like epoxide hydrolases characterized by a conserved His/Tyr ring-opening pair. PMID:21933119

  7. Pseudomonas aeruginosa Cif defines a distinct class of α/β epoxide hydrolases utilizing a His/Tyr ring-opening pair.

    PubMed

    Bahl, Christopher D; Madden, Dean R

    2012-02-01

    The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that secretes a multitude of virulence factors during the course of infection. Among these is Cif, an epoxide hydrolase (EH) that reduces the functional localization of the cystic fibrosis transmembrane conductance regulator in epithelial cells. In addition to being the first reported EH virulence factor, Cif possesses unique sequence deviations from canonical EH motifs. Foremost among these is the substitution of a histidine for the first epoxide ring-opening tyrosine in the active site. To test the functional equivalence of Tyr and His side chains at this position, we have generated the mutant Cif-H177Y. Structural analysis confirms that both the WT His and mutant Tyr side chains can be accommodated without large-scale conformational changes. However, the Tyr mutant is functionally inactive. Based on a detailed analysis of the structure of the Tyr mutant, it appears that Cif's main-chain conformation imposes a functional requirement for a His at this position. Comparison with canonical EH structures reveals additional conformational differences, which are coupled to divergent sequence characteristics. When used to probe the genomes of other opportunistic pathogens, these sequence-structure criteria uncover candidate sequences that appear to form a distinct subfamily of Cif-like epoxide hydrolases characterized by a conserved His/Tyr ring-opening pair.

  8. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

    SciTech Connect

    Duan, Hongying; Yoshimura, Kazunori; Kobayashi, Nobuharu; Sugiyama, Kazuo; Sawada, Jun-ichi; Saito, Yoshiro; Morisseau, Christophe; Hammock, Bruce D.; Akatsuka, Toshitaka

    2012-04-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

  9. Microsomal epoxide hydrolase gene polymorphisms and risk of chronic obstructive pulmonary disease: A comprehensive meta-analysis.

    PubMed

    Li, Hui; Fu, Wei-Ping; Hong, Ze-Hui

    2013-03-01

    Microsomal epoxide hydrolase (EPHX1) is an enzyme involved in the detoxification the products of smoking and is proposed to be a genetic factor for the development of chronic obstructive pulmonary disease (COPD). Two functional polymorphisms of EPHX1, T113C and A139G, have been analyzed in numerous studies to assess the COPD risk attributed to these variants. However, the conclusions were controversial. We performed a comprehensive meta-analysis to clarify these findings. A total of 24 studies comprising 8,259 COPD patients and 42,883 controls were included. The overall results showed that the EPHX1 113 mutant homozygote was significantly associated with an increased risk of COPD (OR, 1.33; 95% CI, 1.06-1.69). The subgroup analyses demonstrated this association in Caucasian individuals (OR, 1.61; 95% CI, 1.12-2.31) but not in Asian individuals. The 139 mutant heterozygote was significantly associated with a decreased risk of COPD in Asian populations (OR, 0.82; 95% CI, 0.68-0.99) but not in Caucasian populations. Pooled analyses revealed that the extremely slow (OR, 1.77; 95% CI, 1.23-2.55) and slow EPHX1 enzyme activity (OR, 1.44; 95% CI, 1.13-1.85) were associated with an increased risk of COPD, while the fast enzyme activity was not associated with a decreased risk of COPD. The stratified analysis demonstrated this association in Caucasian but not in Asian individuals. Furthermore, a modest difference in the risk of COPD was observed between the subgroups by using the cigarette smokers or the non-smokers as controls. A significant correlation between the two functional polymorphisms, T113C and A139G, of the EPHX1 gene and the enzyme activity and the individual's susceptibility to COPD was noted. In addition, the results supported a contribution of EPHX1 to the aetiology of COPD.

  10. Structure activity relationships of cycloalkylamide derivatives as inhibitors of the soluble epoxide hydrolase

    PubMed Central

    Kim, In-Hae; Park, Yong-Gyu; Hammock, Bruce D.; Nishi, Kosuke

    2012-01-01

    Structure activity relationships of cycloalkylamide compounds as inhibitors of human sEH were investigated. When the left side of amide function was modified by a variety of cycloalkanes, at least a C6 like cyclohexane was necessary to yield reasonable inhibition potency on the target enzyme. In compounds with a smaller cycloalkane or with a polar group on the left side of amide function, no inhibition was observed. On the other hand, increased hydrophobicity dramatically improved inhibition potency. Especially, a tetrahydronaphthalene (20) effectively increased the potency. When a series of alkyl or aryl derivatives of cycloalkylamide were investigated to continuously optimize the right side of the amide pharmacophore, a benzyl moiety functionalized with a polar group produced highly potent inhibition. Non-substituted benzyl, alkyl, aryl, or biaryl structure present in the right side of cycloalkylamide function induced a big decrease in inhibition potency. Also, a resulting potent cycloalkylamide (32) showed reasonable physical properties. PMID:21338111

  11. Characterization of the SgcF Epoxide Hydrolase Supporting an (R)-Vicinal Diol Intermediate for Enediyne Antitumor Antibiotic C-1027 Biosynthesis

    PubMed Central

    Lin, Shuangjun; Horsman, Geoffrey P.; Chen, Yihua; Li, Wenli; Shen, Ben

    2009-01-01

    C-1027 is a chromoprotein antitumor antibiotic consisting of an apo-protein and the C-1027 chromophore. The C-1027 chromophore possesses four distinct structural moieties – an enediyne core, a deoxy aminosugar, a benzoxazolinate, and an (S)-3-chloro-5-hydroxy-β-tyrosine – the latter two of which are proposed to be appended to the enediyne core via a convergent biosynthetic strategy. Here we report the in vitro characterization of SgcF, an epoxide hydrolase from the C-1027 biosynthetic gene cluster that catalyzes regio- and stereospecific hydrolysis of styrene oxide, serving as an enediyne core epoxide intermediate mimic, to form a vicinal diol. Abolishment of C-1027 production in the ΔsgcF mutant strain Streptomyces globisporus SB1010 unambiguously establishes that sgcF plays an indispensable role in C-1027 biosynthesis. SgcF efficiently hydrolyzes (S)-styrene oxide, displaying an apparent Km of 0.6 ± 0.1 mM and kcat of 48 ± 1 min−1, via attack at the α-position to exclusively generate the (R)-phenyl vicinal diol, consistent with the stereochemistry of the C-1027 chromophore. These findings support the role of SgcF in the proposed convergent pathway for C-1027 biosynthesis, unveiling an (R)-vicinal diol as a key intermediate. Interestingly, SgcF can also hydrolyze (R)-styrene oxide to afford preferentially the (R)-phenyl vicinal diol via attack at the β-position, albeit with significantly reduced efficiency (apparent Km of 2.0 ± 0.4 mM and kcat = 4.3 ± 0.3 min−1). Although the latter activity unlikely contributes to C-1027 biosynthesis in vivo, such enantioconvergence arising from complementary regioselective hydrolysis of a racemic substrate could be exploited to engineer epoxide hydrolases with improved regio- and/or enantiospecificity. PMID:19856960

  12. Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

    DTIC Science & Technology

    1986-07-01

    carboxylester hydrolases and pancreatic proteinases (3). The choice of organophosphinate candidates for pretreatment agents should be made with these factors...M 4 Q r 000 44 Table 11. Purification of arylester hydrolase Specific Total Total Activity Volume Activity Proteina (Umoles/ Purifi- Fraction (mL...reaction products after the racemic mixture was exposed to arylester hydrolase (EC 3.1.1.2) or proteinases ; chiral-phase capillary gas-liquid

  13. A new phenolic derivative with soluble epoxide hydrolase and nuclear factor-kappaB inhibitory activity from the aqueous extract of Acacia catechu.

    PubMed

    Sun, Ya Nan; Li, Wei; Song, Seok Bean; Yan, Xi Tao; Zhao, Yan; Jo, A Reum; Kang, Jong Seong; Young Ho, Kim

    2016-09-01

    One novel phenolic compound, (4S,5R)-4-(3,4-dihydroxyphenyl)-5-(3-oxobutyl)dihydrofuran-2(3H)-one (1), as well as 12 known compounds (2-13) was obtained from the aqueous extract of Acacia catechu and their chemical structures were determined by spectroscopic analysis. Compounds 8 and 9 exhibited significant soluble epoxide hydrolase (sEH) inhibitory activities with IC50 values of 26.6 ± 0.5 and 24.4 ± 5.6 μM, respectively. Compounds 7-10 showed significant inhibitory effects on TNFα-induced nuclear factor kappa B (NF-κB) transcriptional activity in a dose-dependent manner, with IC50 values ranging from 11.15 to 19.45 μM.

  14. Effect of fungal mycelia on the HPLC-UV and UV-vis spectrophotometric assessment of mycelium-bound epoxide hydrolase using glycidyl phenyl ether.

    PubMed

    Dolcet, Marta M; Torres, Mercè; Canela, Ramon

    2016-06-25

    The use of mycelia as biocatalysts has technical and economic advantages. However, there are several difficulties in obtaining accurate results in mycelium-catalysed reactions. Firstly, sample extraction, indispensable because of the presence of mycelia, can bring into the extract components with a similar structure to that of the analyte of interest; secondly, mycelia can influence the recovery of the analyte. We prepared calibration standards of 3-phenoxy-1,2-propanediol (PPD) in the pure solvent and in the presence of mycelia (spiked before or after extraction) from five fungi (Aspergillus niger, Aspergillus tubingensis, Penicillium aurantiogriseum, Penicillium sp. and Aspergillus terreus). The quantification of PPD was carried out by HPLC-UV and UV-vis spectrophotometry. The manuscript shows that the last method is as accurate as the HPLC method. However, the colorimetric method led to a higher data throughput, which allowed the study of more samples in a shorter time. Matrix effects were evaluated visually from the plotted calibration data and statistically by simultaneously comparing the intercept and slope of calibration curves performed with solvent, post-extraction spiked standards and pre-extraction spiked standards. Significant differences were found between the post- and pre-extraction spiked matrix-matched functions. Pre-extraction spiked matrix-matched functions based on A. tubingensis mycelia, selected as the reference, were validated and used to compensate for low recoveries. These validated functions were successfully applied to the quantification of PPD achieved during the hydrolysis of glycidyl phenyl ether by mycelium-bound epoxide hydrolases and equivalent hydrolysis yields were determined by HPLC-UV and UV-vis spectrophotometry. This study may serve as starting point to implement matrix effects evaluation when mycelium-bound epoxide hydrolases are studied.

  15. Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.).

    PubMed

    Deshpande, Ashish B; Chidley, Hemangi G; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

    2017-06-01

    Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango.

  16. Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis.

    PubMed

    Lindberg, Diana; Ahmad, Shabbir; Widersten, Mikael

    2010-03-15

    Epoxide hydrolase, StEH1, shows hysteretic behavior in the catalyzed hydrolysis of trans-2-methylstyrene oxide (2-MeSO)(1). Linkage between protein structure dynamics and catalytic function was probed in mutant enzymes in which surface-located salt-bridging residues were substituted. Salt-bridges at the interface of the alpha/beta-hydrolase fold core and lid domains, as well as between residues in the lid domain, between Lys(179)-Asp(202), Glu(215)-Arg(41) and Arg(236)-Glu(165) were disrupted by mutations, K179Q, E215Q, R236K and R236Q. All mutants displayed enzyme activity with styrene oxide (SO) and 2-MeSO when assayed at 30 degrees C. Disruption of salt-bridges altered the rates for isomerization between distinct Michaelis complexes, with (1R,2R)-2-MeSO as substrate, presumably as a result of increased dynamics of involved protein segments. Another indication of increased flexibility was a lowered thermostability in all mutants. We propose that the alterations to regioselectivity in these mutants derive from an increased mobility in protein segments otherwise stabilized by salt bridging interactions.

  17. A model of sensitivity: 1,3-butadiene increases mutant frequencies and genomic damage in mice lacking a functional microsomal epoxide hydrolase gene.

    PubMed

    Wickliffe, Jeffrey K; Ammenheuser, Marinel M; Salazar, James J; Abdel-Rahman, Sherif Z; Hastings-Smith, Darlene A; Postlethwait, Edward M; Lloyd, R Stephen; Ward, Jonathan B

    2003-01-01

    The specific role that polymorphisms in xenobiotic metabolizing enzymes play in modulating sensitivity to 1,3-butadiene (BD) genotoxicity has been relatively unexplored. The enzyme microsomal epoxide hydrolase (mEH) is important in detoxifying the mutagenic epoxides of BD (butadiene monoepoxide [BDO], butadiene diepoxide [BDO(2)]). Polymorphisms in the human mEH gene appear to affect the function of the enzyme. We exposed mice with normal mEH activity (WT) and knockout mice without mEH activity (KO) to 20 ppm BD (inhalation) or 30 mg/kg BDO(2) (intraperitoneal [IP] injection). We then compared Hprt mutant frequencies (MFs) among these groups. KO mice exposed to BD exhibited a significant (P < 0.05) 12.4-fold increase in MF over controls and a significant 5.4-fold increase in MF over exposed WT mice. Additionally, KO mice exposed to BDO(2) exhibited a significant 4.5-fold increase in MF over controls and a significant 1.7-fold increase in MF over exposed WT mice. We also compared genomic damage in WT and KO mice (comet tail moment) following IP exposure to 3 mg/kg and 30 mg/kg BDO(2). KO mice exposed to 3 mg/kg exhibited significantly more DNA damage than controls (7.5-12.1-fold increase) and exposed WT mice (3 mg/kg; 4.8-fold increase). KO mice exposed to 30 mg/kg BDO(2) exhibited significantly more DNA damage than all other groups (2.3-27.9-fold increase). Correlation analysis indicated that a significant, positive relationship (r(2) = 0.92) exists between comet-measured damage and Hprt MFs. The lack of mEH activity increases the genetic sensitivity of mice exposed to BD and BDO(2). This model should facilitate a mechanistic understanding of the observed variation in human genetic sensitivity following exposure to BD. Copyright 2003 Wiley-Liss, Inc.

  18. Effect of CYP2E1 gene deletion in mice on expression of microsomal epoxide hydrolase in response to VCD exposure.

    PubMed

    Keating, Aileen F; Rajapaksa, Kathila S; Sipes, I Glenn; Hoyer, Patricia B

    2008-10-01

    Females are born with a finite number of primordial follicles. 4-Vinylcyclohexene diepoxide (VCD) is a metabolite formed by epoxidation of 4-vinylcyclohexene (VCH) via its two monoepoxides 1,2- and 7,8-4-vinylcyclohexene monoepoxide (VCM). VCD specifically destroys small preantral (primordial and small primary) follicles in the rodent ovary. The phase I enzyme, cytochrome P450 isoform 2E1 (CYP2E1) is involved in ovarian metabolism of VCM to VCD. Further, microsomal epoxide hydrolase (mEH) can detoxify VCD to an inactive tetrol (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane). This study evaluated the effects of VCD-induced ovotoxicity on mEH in CYP2E1+/+ and -/- mice (129S(1)/SvImJ background strain) using a postnatal day 4 mouse whole ovary culture system. The hypothesis of our study is that there is a relationship between CYP2E1 and mEH gene expression in the mouse ovary. Relative to control, VCD exposure caused follicle loss (p < 0.05) in ovaries from both genotypes; however, after 15 days, this loss was greater (p < 0.05) in CYP2E1+/+ ovaries. In a time course (2-15 days), relative to control, VCD (5 microM) caused an increase (p < 0.05) in mEH mRNA by 0.5-fold (day 10) and 1.84-fold (day 15) in CYP2E1-/- but not +/+ ovaries. 7,12-Dimethylbenz[a]anthracene (DMBA) also destroys ovarian follicles but, unlike VCD, is bioactivated by mEH to an ovotoxic 3,4-diol-1,2-epoxide metabolite. Incubation of ovaries in increasing concentrations of DMBA (0.5-1 microM, 15 days) resulted in greater (p < 0.05) follicle loss in CYP2E1-/-, relative to +/+ ovaries. With greater mEH (CYP2E1-/-), increased follicle loss with DMBA (bioactivation) and decreased follicle loss with VCD (detoxification) support that ovarian expression of CYP2E1 and mEH may be linked.

  19. Comparative metabolism of methacrylonitrile and acrylonitrile to cyanide using cytochrome P4502E1 and microsomal epoxide hydrolase-null mice

    SciTech Connect

    El Hadri, L.; Chanas, B.; Ghanayem, B.I. . E-mail: ghanayem@niehs.nih.gov

    2005-06-01

    Methacrylonitrile (MAN) and acrylonitrile (AN) are metabolized via glutathione (GSH) conjugation or epoxide formation. We have recently shown that CYP2E1 is essential for AN epoxidation and subsequent cyanide liberation. Current studies were designed to compare the enzymatic basis of MAN vs. AN metabolism to cyanide using wild-type (WT), CYP2E1-, and mEH-null mice. Mice received a single gavage dose of 0.047, 0.095, 0.19, or 0.38 mmol/kg of MAN or AN, and blood cyanide was measured at 1 or 3 h later. Blood cyanide levels in WT mice treated with AN or MAN were dose and time dependent. At equimolar doses, significantly higher levels of cyanide were detected in the blood of MAN- vs. AN-treated mice. Further, while significant reduction in blood cyanide levels occurred in MAN-treated CYP2E1-null vs. WT mice, AN metabolism to cyanide was largely abolished in CYP2E1-null mice. Pretreatment of mice with 1-aminobenzotriazole (ABT, CYP inhibitor) demonstrated that CYPs other than CYP2E1 also contribute to MAN metabolism to cyanide. Blood cyanide levels in mEH-null mice treated with aliphatic nitriles are generally lower than levels in similarly treated WT mice. Western blot analysis showed that expression of sEH was greater in male vs. female mice. The role of various epoxide hydrolases (EHs) in the production of cyanide from aliphatic nitriles is apparently structure and dose dependent. Regardless of genotype, significantly higher levels of cyanide were measured in the blood of male vs. female mice treated with MAN or AN. In conclusion, these data showed that (1) at equimolar doses, higher blood cyanide levels were detected in mice treated with MAN vs. AN; (2) while CYP2E1 is the only enzyme responsible for AN metabolism to cyanide, other CYPs also contribute to MAN metabolism; and (3) significantly higher levels of cyanide were measured in the blood of male vs. female treated with either nitrile. Higher blood cyanide levels in male vs. female mice and in MAN- vs. AN

  20. Coinduction of cytochrome P450IIE1, glutathione S-transferases and microsomal epoxide hydrolase by nitrogen- and sulfur-containing heterocycles in rat liver

    SciTech Connect

    Kim, S.G.; Novak, R.F. )

    1991-03-15

    The effects of thiazole, pyrazine, pyridazine and pyrimidine on cytochrome P450IIE1, the glutathione S-transferases (GSTs) and microsomal epoxide hydrolase (mEH) have been examined at the molecular level. Administration of each of these compounds to rats was found to elevate hepatic P450IIE1, the GSTs, and mEH simultaneously, as evidenced by catalytic activities, SDS-PAGE and immunoblot analyses. Hepatic tissue was obtained at 24, 48 and 72 h during the treatment regimen. RNA and LiCl precipitated from hepatic tissue homogenized in guanidinium thiocyanate and poly(A){sup +} RNA was isolated using oligo(dT) cellulose. Slot and Northern blot analyses of poly(A){sup +} RNA isolated from rats during the 3 d treatment regimen revealed an {approximately}4 to 5-fold decrease in P450IIE1 mRNA at 24 h after treatment with a slight increase noted for 48 and 72 h relative to untreated animals. In contrast, an {approximately}8 to 14-fold increase in GST {alpha}-class mRNA and an {approximately}17- to 20-fold increase in mEH mRNA was monitored at 48 to 72 h as compared to untreated animals. These results revealed that these heterocyclic compounds induce both Phase 1 and Phase 2 drug metabolizing enzymes simultaneously through different molecular mechanisms.

  1. Intronic DNA elements regulate Nrf-2 chemical responsiveness of the human microsomal epoxide hydrolase gene (EPHX1) through a far upstream alternative promoter

    PubMed Central

    Su, Shengzhong; Yang, Xi; Omiecinski, Curtis J.

    2014-01-01

    In humans, microsomal epoxide hydrolase (mEH) contributes important biological functions that underlie both detoxification and bioactivation fates arising from exposures to foreign chemicals. Previously, we discovered that human mEH gene transcription is initiated from alternative promoters. The respective transcripts are programmed with tissue specificity and the upstream E1b promoter contributes predominantly to mEH expression. The results presented demonstrate that exposures to the Nrf2 activators, sulforaphane (SFN) and tert-butylhydroquinone (tBHQ), markedly activate E1b transcription in human lung and liver cells. Genomic analyses identified two major DNase I hypersensitive regions (HS-1 and HS-2) within the ~15 kb intervening sequence separating E1b from the downstream E1 promoter. In BEAS-2B cells, the Nrf2 effectors, SFN and tBHQ, selectively activated the more distal HS-2 through an antioxidant-response element (ARE). An activator protein 1/12-O-tetradecanoylphorbol-13-acetate interaction was further identified within the HS-2 enhancer that functioned to additionally contribute to ARE-mediated induction responsiveness of the E1b promoter. The results demonstrate that ARE modulation, integrated with additional transcriptional complexes, regulates the tissue-specific expression of mEH and that these processes likely coordinate both the protective and bioactivation functions contributed by mEH activities in human tissues. PMID:24704207

  2. N-3 Polyunsaturated Fatty Acids Decrease the Protein Expression of Soluble Epoxide Hydrolase via Oxidative Stress-Induced P38 Kinase in Rat Endothelial Cells.

    PubMed

    Okada, Takashi; Morino, Katsutaro; Nakagawa, Fumiyuki; Tawa, Masashi; Kondo, Keiko; Sekine, Osamu; Imamura, Takeshi; Okamura, Tomio; Ugi, Satoshi; Maegawa, Hiroshi

    2017-06-24

    N-3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n-3 PUFAs, increased in n-3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n-3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n-3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n-3 PUFAs may contribute to their cardio-protective effect.

  3. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases

    SciTech Connect

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; Brunecky, Roman; Xu, Qi; Lunin, Vladimir V.; Brady, John W.; Crowley, Michael F.; Himmel, Michael E.; Bomble, Yannick J.

    2016-02-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. As a result, the theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.

  4. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases.

    PubMed

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; Brunecky, Roman; Xu, Qi; Lunin, Vladimir V; Brady, John W; Crowley, Michael F; Himmel, Michael E; Bomble, Yannick J

    2016-03-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. The theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.

  5. Expression of ovarian microsomal epoxide hydrolase and glutathione S-transferase during onset of VCD-induced ovotoxicity in B6C3F(1) mice.

    PubMed

    Keating, Aileen F; Sipes, I Glenn; Hoyer, Patricia B

    2008-07-01

    4-vinylcyclohexene diepoxide (VCD) specifically destroys small pre-antral follicles in the rodent ovary. VCD can be detoxified to an inactive tetrol by microsomal epoxide hydrolase (mEH), or by conjugation to glutathione (GSH) by glutathione S-transferase (GST). Formation of VCD-GSH adducts in the mouse ovary 4 h after VCD exposure (0.57 mmol/kg/day) has been demonstrated. Because the mouse ovary expresses both mEH and GST, expression of mEH and GST pi and mu during a time-course of VCD-induced ovotoxicity was evaluated in a neonatal mouse ovarian culture system. Ovaries from postnatal day 4 (PND4) B6C3F(1) mice were incubated with VCD (15 microM) for 2, 4, 6, 8, 10, 12, or 15 days. Following incubation, ovaries were histologically evaluated, or assessed for mRNA or protein expression. VCD did not cause follicle loss (p>0.05) on days 2, 4, or 6 of culture. At days 8, 10, 12, and 15, VCD reduced (p<0.05) both primordial and primary follicle numbers. Increased (p<0.05) expression of mEH, GST pi and GST mu mRNA was detected after 4 days of VCD exposure. This expression was reduced on days 6 and 8, when follicle loss was underway, but increased (p<0.05) after 10 days of exposure. mEH and GST pi proteins were elevated (p<0.05) following 8 days of VCD-exposure however there was no increase in GST mu protein. These findings suggest that with continuous exposure to VCD, increased expression of detoxification enzymes may participate in retarding the onset of follicle loss, but that this loss cannot ultimately be prevented.

  6. Expression of ovarian microsomal epoxide hydrolase and glutathione S-transferase during onset of VCD-induced ovotoxicity in B6C3F{sub 1} mice

    SciTech Connect

    Keating, Aileen F.; Sipes, I. Glenn; Hoyer, Patricia B.

    2008-07-01

    4-vinylcyclohexene diepoxide (VCD) specifically destroys small pre-antral follicles in the rodent ovary. VCD can be detoxified to an inactive tetrol by microsomal epoxide hydrolase (mEH), or by conjugation to glutathione (GSH) by glutathione S-transferase (GST). Formation of VCD-GSH adducts in the mouse ovary 4 h after VCD exposure (0.57 mmol/kg/day) has been demonstrated. Because the mouse ovary expresses both mEH and GST, expression of mEH and GST pi and mu during a time-course of VCD-induced ovotoxicity was evaluated in a neonatal mouse ovarian culture system. Ovaries from postnatal day 4 (PND4) B6C3F{sub 1} mice were incubated with VCD (15 {mu}M) for 2, 4, 6, 8, 10, 12, or 15 days. Following incubation, ovaries were histologically evaluated, or assessed for mRNA or protein expression. VCD did not cause follicle loss (p > 0.05) on days 2, 4, or 6 of culture. At days 8, 10, 12, and 15, VCD reduced (p < 0.05) both primordial and primary follicle numbers. Increased (p < 0.05) expression of mEH, GST pi and GST mu mRNA was detected after 4 days of VCD exposure. This expression was reduced on days 6 and 8, when follicle loss was underway, but increased (p < 0.05) after 10 days of exposure. mEH and GST pi proteins were elevated (p < 0.05) following 8 days of VCD-exposure however there was no increase in GST mu protein. These findings suggest that with continuous exposure to VCD, increased expression of detoxification enzymes may participate in retarding the onset of follicle loss, but that this loss cannot ultimately be prevented.

  7. Effect of epoxide hydrolase and glutathione S-tranferase genotypes on the induction of micronuclei and DNA damage by styrene-7,8-oxide in vitro.

    PubMed

    Laffon, Blanca; Pérez-Cadahía, Beatriz; Pásaro, Eduardo; Méndez, Josefina

    2003-04-20

    Styrene is one of the most important organic chemicals used worldwide. Its main metabolite, styrene-7,8-oxide (SO), is considered responsible for the genotoxic effects associated with exposure to styrene. SO is detoxified by hydrolysis catalyzed by epoxide hydrolase (EH), or, to a minor extent, by conjugation mediated by glutathione S-transferases (GSTs). The purpose of the present study was to investigate whether EH (exons 3 and 4), GSTP1 (exons 5 and 6), GSTM1 and GSTT1 polymorphisms have any influence on the genotoxicity of SO in human leukocytes. Peripheral leukocytes from 30 healthy donors were exposed to SO (50 and 200 micro M) and genotoxicity was evaluated by means of the micronucleus (MN) test and alkaline comet assay, using 1% DMSO as solvent control. When EH genotypes were classified in low, medium, and high with respect to the expected EH activity, an increase in induced comet tail length was observed with decreasing EH activity in SO-exposed cells. An increase was seen in induced MN frequency in EH low-activity donors. These findings are consistent with the detoxifying activity of this enzyme. In addition, increases in MN frequencies for GSTP1 *A/*B and *A/*C genotypes with regard to the wild-type homozygous *A/*A genotype were detected. This may be due to a low detoxifying activity as a consequence of altered SO affinity of the variant protein, but must be confirmed using homozygote variant individuals, not included in this study. No clear results were obtained for GSTM1 or GSTT1 genotypes, even when performing the analysis after grouping individuals with the same expected EH activity, probably due to the minor role that glutathione conjugation plays in styrene metabolism. The present in vitro findings using human leukocytes suggest that polymorphisms in EH, and, to a lesser extent, in GSTP1, may influence induction of cytogenetic and DNA damage by SO.

  8. Genetic susceptibility to chronic obstructive pulmonary disease in Koreans: combined analysis of polymorphic genotypes for microsomal epoxide hydrolase and glutathione S-transferase M1 and T1

    PubMed Central

    Yim, J.; Park, G. Y.; Lee, C.; Kim, Y. W.; Han, S. K.; Shim, Y.; Yoo, C.

    2000-01-01

    BACKGROUND—Although smoking is the major causal factor in the development of chronic obstructive pulmonary disease (COPD), only 10-20% of chronic heavy cigarette smokers develop symptomatic COPD which suggests the presence of genetic susceptibility. This genetic susceptibility to COPD might depend on variations in enzyme activities that detoxify cigarette smoke products such as microsomal epoxide hydrolase (mEPHX) and glutathione-S transferase (GST). As there is increasing evidence that several genes influence the development of COPD, multiple gene polymorphisms should be investigated to find out the genetic susceptibility to COPD.
METHODS—The genotypes of 83 patients with COPD and 76 healthy smoking control subjects were determined by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (PCR-RFLP) for the mEPHX gene, and multiplex PCR for GST M1 and GST T1 genes. The frequencies of polymorphic genotypes of mEPHX, GST M1, and GST T1 genes were compared both individually and in combination in patients with COPD and healthy smokers.
RESULTS—No differences were observed in the frequency of polymorphic genotypes in exons 3 and 4 of mEPHX, GST M1, and GST T1 genes between patients with COPD and healthy smokers. The frequencies of any combination of these genotypes also showed no differences between the COPD group and the control group.
CONCLUSIONS—Genetic polymorphisms in mEPHX, GST M1, and GST T1 genes are not associated with the development of COPD in Koreans.

 PMID:10639528

  9. Early postnatal treatment with soluble epoxide hydrolase inhibitor or 15-deoxy-Δ(12,14)-prostagandin J2 prevents prenatal dexamethasone and postnatal high saturated fat diet induced programmed hypertension in adult rat offspring.

    PubMed

    Lu, Pei-Chen; Sheen, Jiunn-Ming; Yu, Hong-Ren; Lin, Yu-Ju; Chen, Chih-Cheng; Tiao, Mao-Meng; Tsai, Ching-Chou; Huang, Li-Tung; Tain, You-Lin

    2016-07-01

    Prenatal dexamethasone (DEX) exposure, postnatal high-fat (HF) intake, and arachidonic acid pathway are closely related to hypertension. We tested whether a soluble epoxide hydrolase (SEH) inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) or 15-deoxy-Δ(12,14)-prostagandin J2 (15dPGJ2) therapy can rescue programmed hypertension in the DEX+HF two-hit model. Four groups of Sprague Dawley rats were studied: control, DEX+HF, AUDA, and 15dPGJ2. Dexamethasone (0.1mg/kg body weight) was intraperitoneally administered to pregnant rats from gestational day 16-22. Male offspring received high-fat diet (D12331, Research Diets) from weaning to 4 months of age. In AUDA group, mother rats received 25mg/L in drinking water during lactation. In the 15dPGJ2 group, male offspring received 15dPGJ2 1.5mg/kg BW by subcutaneous injection once daily for 1 week after birth. We found postnatal HF diet aggravated prenatal DEX-induced programmed hypertension, which was similarly prevented by early treatment with AUDA or 15dPGJ2. The beneficial effects of AUDA and 15d-PGJ2 therapy include inhibition of SEH, increases of renal angiotensin converting enzyme-2 (ACE2) and angiotensin II type 2 receptor (AT2R) protein levels, and restoration of nitric oxide bioavailability. Better understanding of the impact of arachidonic acid pathway in the two-hit model will help prevent programmed hypertension in children exposed to corticosteroids and postnatal HF intake.

  10. Transcription of the Human Microsomal Epoxide Hydrolase Gene (EPHX1) Is Regulated by PARP-1 and Histone H1.2. Association with Sodium-Dependent Bile Acid Transport.

    PubMed

    Peng, Hui; Zhu, Qin-shi; Zhong, Shuping; Levy, Daniel

    2015-01-01

    Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes. These compounds are involved in cholesterol homeostasis, lipid digestion, excretion of xenobiotics and the regulation of several nuclear receptors and signaling transduction pathways. Previous studies have demonstrated the critical role of GATA-4, a C/EBPα-NF/Y complex and an HNF-4α/CAR/RXR/PSF complex in the transcriptional regulation of the mEH gene (EPHX1). Studies also identified heterozygous mutations in human EPHX1 that resulted in a 95% decrease in mEH expression levels which was associated with a decrease in bile acid transport and severe hypercholanemia. In the present investigation we demonstrate that EPHX1 transcription is significantly inhibited by two heterozygous mutations observed in the Old Order Amish population that present numerous hypercholanemic subjects in the absence of liver damage suggesting a defect in bile acid transport into the hepatocyte. The identity of the regulatory proteins binding to these sites, established using biotinylated oligonucleotides in conjunction with mass spectrometry was shown to be poly(ADP-ribose)polymerase-1 (PARP-1) bound to the EPHX1 proximal promoter and a linker histone complex, H1.2/Aly, bound to a regulatory intron 1 site. These sites exhibited 71% homology and may represent potential nucleosome positioning domains. The high frequency of the H1.2 site polymorphism in the Amish population results in a potential genetic predisposition to hypercholanemia and in conjunction with our previous studies, further supports the critical role of mEH in mediating bile acid transport into hepatocytes.

  11. Vascular Endothelial Over-Expression of Human Soluble Epoxide Hydrolase (Tie2-sEH Tr) Attenuates Coronary Reactive Hyperemia in Mice: Role of Oxylipins and ω-Hydroxylases

    PubMed Central

    Zeldin, Darryl C.; Morisseau, Christophe; Falck, John R.

    2017-01-01

    Cytochromes P450 metabolize arachidonic acid (AA) into two vasoactive oxylipins with opposing biologic effects: epoxyeicosatrienoic acids (EETs) and omega-(ω)-terminal hydroxyeicosatetraenoic acids (HETEs). EETs have numerous beneficial physiological effects, including vasodilation and protection against ischemia/reperfusion injury, whereas ω-terminal HETEs induce vasoconstriction and vascular dysfunction. We evaluated the effect of these oxylipins on post-ischemic vasodilation known as coronary reactive hyperemia (CRH). CRH prevents the potential harm associated with transient ischemia. The beneficial effects of EETs are reduced after their hydrolysis to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). ω-terminal HETEs are formed by ω-hydroxylase family members. The relationship among endothelial over-expression of sEH (Tie2-sEH Tr), the changes in oxylipins it may produce, the pharmacologic inhibition of ω-hydroxylases, activation of PPARγ, and CRH response to a brief ischemia is not known. We hypothesized that CRH is attenuated in isolated mouse hearts with endothelial sEH over-expression through modulation of oxylipin profiles, whereas both inhibition of ω-hydroxylases and activation of PPARγ enhance CRH. Compared to WT mice, Tie2-sEH Tr mice had decreased CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05), whereas inhibition of ω-hydroxylases increased these same CRH parameters in Tie2-sEH Tr mice. Inhibition of sEH with t-AUCB reversed the decreased CRH in Tie2-sEH Tr mice. Endothelial over-expression of sEH significantly changed oxylipin profiles, including decreases in DHETs, mid-chain HETEs, and prostaglandins (P < 0.05). Treatment with rosiglitazone, PPARγ-agonist, enhanced CRH (P < 0.05) in both Tie2-sEH Tr and wild type (WT) mice. These data demonstrate that endothelial over-expression of sEH (through changing the oxylipin profiles) attenuates CRH, whereas inhibition of

  12. The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology.

    PubMed Central

    Friedberg, T; Holler, R; Löllmann, B; Arand, M; Oesch, F

    1996-01-01

    Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786-5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial huminal orientation, displayed rather

  13. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases

    DOE PAGES

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; ...

    2016-02-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the productmore » inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. As a result, the theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.« less

  14. Modulation by salt intake of the vascular response mediated through adenosine A(2A) receptor: role of CYP epoxygenase and soluble epoxide hydrolase.

    PubMed

    Nayeem, Mohammed A; Zeldin, Darryl C; Boegehold, Matthew A; Morisseau, Christophe; Marowsky, Anne; Ponnoth, Dovenia S; Roush, Kevin P; Falck, John R

    2010-07-01

    High-salt intake can change the effect of adenosine on arterial tone in mice. The aim of this study was to clarify the mechanism by which this occurs. Using aortas from mice fed a 4% NaCl (HS) or 0.45% NaCl (NS) diet for 4-5 wks, concentration-response curves for ACh, 5'-N-ethylcarboxamidoadenosine (NECA; adenosine analog) and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate [CGS-21680; A(2A) adenosine receptor (A(2A) AR) agonist] were obtained with N(omega)-nitro-L-arginine methyl ester (L-NAME; nitric oxide inhibitor, 10(-4) M), methylsulfonyl-propargyloxyphenylhexanamide [MS-PPOH; a CYP (cytochrome P-450) epoxygenase blocker, 10(-5) M including CYP2J2], 12-(3-adamantan-1-yl-ureido)dodecanoic acid [AUDA; soluble epoxide hydrolase (sEH) blocker, 10(-5) M], dibromo-dodecenyl-methylsulfimide [DDMS; CYP omega-hydroxylase (CYP4A blocker), 10(-5) M], glibenclamide (K(ATP) channel blocker; 10(-5) M) and 5-hydroxydecanoate (5-HD; mitochondrial-K(ATP) channel blocker, 10(-4) M). HS dose response to ACh (10(-7) - 10(-5) M) was not different from NS (P > 0.05). Relaxation to 10(-6) M NECA was greater in the HS group (28.4 +/- 3.9%) than in the NS group (4.1 +/- 2.3%). Relaxation to 10(-6) M CGS-21680 was also greater in HS (27.9 +/- 4.5%) than in NS (4.9 +/- 2.2%). L-NAME was able to block the dose response of ACh (10(-7) - 10(-5) M) equally in both HS and NS (P > 0.05), whereas L-NAME did not block CGS-21680-induced response in HS. In HS the CGS-21680 response was greatly reduced by MS-PPOH (to 4.7 +/- 2.0%) and 5-HD (to 8.9 +/- 2.2%), and also abolished by glibenclamide (-1.0 +/- 5.9%). In NS, the CGS-21680 response was increased by AUDA (to 26.3 +/- 3.4%) and DDMS (to 27.2 +/- 3.0%). Compared with NS, HS vessels showed increased CYP2J2 and A(2A) AR expression (46 and 74% higher, respectively) but decreased sEH, CYP4A, and A(1) AR expression (75, 30, and 55% lower, respectively). These data suggest that in mice fed NS-containing diet

  15. Modulation by salt intake of the vascular response mediated through adenosine A2A receptor: role of CYP epoxygenase and soluble epoxide hydrolase

    PubMed Central

    Zeldin, Darryl C.; Boegehold, Matthew A.; Morisseau, Christophe; Marowsky, Anne; Ponnoth, Dovenia S.; Roush, Kevin P.; Falck, John R.

    2010-01-01

    High-salt intake can change the effect of adenosine on arterial tone in mice. The aim of this study was to clarify the mechanism by which this occurs. Using aortas from mice fed a 4% NaCl (HS) or 0.45% NaCl (NS) diet for 4–5 wks, concentration-response curves for ACh, 5′-N-ethylcarboxamidoadenosine (NECA; adenosine analog) and 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate [CGS-21680; A2A adenosine receptor (A2A AR) agonist] were obtained with Nω-nitro-l-arginine methyl ester (l-NAME; nitric oxide inhibitor, 10−4 M), methylsulfonyl-propargyloxyphenylhexanamide [MS-PPOH; a CYP (cytochrome P-450) epoxygenase blocker, 10−5 M including CYP2J2], 12-(3-adamantan-1-yl-ureido)dodecanoic acid [AUDA; soluble epoxide hydrolase (sEH) blocker, 10−5 M], dibromo-dodecenyl-methylsulfimide [DDMS; CYP ω-hydroxylase (CYP4A blocker), 10−5 M], glibenclamide (KATP channel blocker; 10−5 M) and 5-hydroxydecanoate (5-HD; mitochondrial-KATP channel blocker, 10−4 M). HS dose response to ACh (10−7 − 10−5 M) was not different from NS (P > 0.05). Relaxation to 10−6 M NECA was greater in the HS group (28.4 ± 3.9%) than in the NS group (4.1 ± 2.3%). Relaxation to 10−6 M CGS-21680 was also greater in HS (27.9 ± 4.5%) than in NS (4.9 ± 2.2%). l-NAME was able to block the dose response of ACh (10−7 − 10−5 M) equally in both HS and NS (P > 0.05), whereas l-NAME did not block CGS-21680-induced response in HS. In HS the CGS-21680 response was greatly reduced by MS-PPOH (to 4.7 ± 2.0%) and 5-HD (to 8.9 ± 2.2%), and also abolished by glibenclamide (−1.0 ± 5.9%). In NS, the CGS-21680 response was increased by AUDA (to 26.3 ± 3.4%) and DDMS (to 27.2 ± 3.0%). Compared with NS, HS vessels showed increased CYP2J2 and A2A AR expression (46 and 74% higher, respectively) but decreased sEH, CYP4A, and A1 AR expression (75, 30, and 55% lower, respectively). These data suggest that in mice fed NS-containing diet, upregulation of

  16. The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function: IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE.

    PubMed

    Chiba, Takahito; Thomas, Christopher P; Calcutt, M Wade; Boeglin, William E; O'Donnell, Valerie B; Brash, Alan R

    2016-07-08

    Creation of an intact skin water barrier, a prerequisite for life on dry land, requires the lipoxygenase-catalyzed oxidation of the essential fatty acid linoleate, which is esterified to the ω-hydroxyl of an epidermis-specific ceramide. Oxidation of the linoleate moiety by lipoxygenases is proposed to facilitate enzymatic cleavage of the ester bond, releasing free ω-hydroxyceramide for covalent binding to protein, thus forming the corneocyte lipid envelope, a key component of the epidermal barrier. Herein, we report the transformations of esterified linoleate proceed beyond the initial steps of oxidation and epoxyalcohol synthesis catalyzed by the consecutive actions of 12R-LOX and epidermal LOX3. The major end product in human and porcine epidermis is a trihydroxy derivative, formed with a specificity that implicates participation of an epoxide hydrolase in converting epoxyalcohol to triol. Of the 16 possible triols arising from hydrolysis of 9,10-epoxy-13-hydroxy-octadecenoates, using LC-MS and chiral analyses, we identify and quantify specifically 9R,10S,13R-trihydroxy-11E-octadecenoate as the single major triol esterified in porcine epidermis and the same isomer with lesser amounts of its 10R diastereomer in human epidermis. The 9R,10S,13R-triol is formed by SN2 hydrolysis of the 9R,10R-epoxy-13R-hydroxy-octadecenoate product of the LOX enzymes, a reaction specificity characteristic of epoxide hydrolase. The high polarity of triol over the primary linoleate products enhances the concept that the oxidations disrupt corneocyte membrane lipids, promoting release of free ω-hydroxyceramide for covalent binding to protein and sealing of the waterproof barrier. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Computational insights into function and inhibition of fatty acid amide hydrolase.

    PubMed

    Palermo, Giulia; Rothlisberger, Ursula; Cavalli, Andrea; De Vivo, Marco

    2015-02-16

    The Fatty Acid Amide Hydrolase (FAAH) enzyme is a membrane-bound serine hydrolase responsible for the deactivating hydrolysis of a family of naturally occurring fatty acid amides. FAAH is a critical enzyme of the endocannabinoid system, being mainly responsible for regulating the level of its main cannabinoid substrate anandamide. For this reason, pharmacological inhibition of FAAH, which increases the level of endogenous anandamide, is a promising strategy to cure a variety of diseases including pain, inflammation, and cancer. Much structural, mutagenesis, and kinetic data on FAAH has been generated over the last couple of decades. This has prompted several informative computational investigations to elucidate, at the atomic-level, mechanistic details on catalysis and inhibition of this pharmaceutically relevant enzyme. Here, we review how these computational studies - based on classical molecular dynamics, full quantum mechanics, and hybrid QM/MM methods - have clarified the binding and reactivity of some relevant substrates and inhibitors of FAAH. We also discuss the experimental implications of these computational insights, which have provided a thoughtful elucidation of the complex physical and chemical steps of the enzymatic mechanism of FAAH. Finally, we discuss how computations have been helpful for building structure-activity relationships of potent FAAH inhibitors. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Behavioral effects of fatty acid amide hydrolase inhibition on morphine withdrawal symptoms.

    PubMed

    Shahidi, Siamak; Hasanein, Parisa

    2011-08-10

    Chronic morphine exposure causes tolerance and dependence. The cessation of morphine consumption induces a withdrawal syndrome that may involve cannabinoids and is characterized by undesirable psychological and physical signs. The present study examined whether augmentation of the endocannabinoid system by inhibition of fatty acid amide hydrolase could suppress the morphine withdrawal syndrome in morphine-addicted rats. Morphine dependency was induced by 7 consecutive days of morphine injection. The morphine-addicted rats received URB597 (1, 0.5, 0.3, 0.1, 0.03 mg/kg), a fatty acid amide hydrolase inhibitor, before the precipitation of morphine withdrawal syndromes by naloxone. Withdrawal symptoms including jumping, teeth chattering, paw tremor, wet dog shakes, face grooming, penis licking, standing, rearing, sniffing and percent of weight loss were recorded during 30 min after naloxone injection. The results showed that the morphine withdrawal precipitated rats had significantly more withdrawal symptoms than naive control rats and the administration of URB597 (all doses except 0.03 mg/kg) reduced most of the morphine withdrawal symptoms. We conclude that the administration of URB597 modulated morphine withdrawal symptoms. This finding shows that endocannabinoids interact with the opioid system during the morphine withdrawal period and that potentiation of the endogenous cannabinoid system by URB597 may be a new target strategy for the management of morphine addiction. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. The Molecular Basis for Dual Fatty Acid Amide Hydrolase (FAAH)/Cyclooxygenase (COX) Inhibition.

    PubMed

    Palermo, Giulia; Favia, Angelo D; Convertino, Marino; De Vivo, Marco

    2016-06-20

    The design of multitarget-directed ligands is a promising strategy for discovering innovative drugs. Here, we report a mechanistic study that clarifies key aspects of the dual inhibition of the fatty acid amide hydrolase (FAAH) and the cyclooxygenase (COX) enzymes by a new multitarget-directed ligand named ARN2508 (2-[3-fluoro-4-[3-(hexylcarbamoyloxy)phenyl]phenyl]propanoic acid). This potent dual inhibitor combines, in a single scaffold, the pharmacophoric elements often needed to block FAAH and COX, that is, a carbamate moiety and the 2-arylpropionic acid functionality, respectively. Molecular modeling and molecular dynamics simulations suggest that ARN2508 uses a noncovalent mechanism of inhibition to block COXs, while inhibiting FAAH via the acetylation of the catalytic Ser241, in line with previous experimental evidence for covalent FAAH inhibition. This study proposes the molecular basis for the dual FAAH/COX inhibition by this novel hybrid scaffold, stimulating further experimental studies and offering new insights for the rational design of novel anti-inflammatory agents that simultaneously act on FAAH and COX.

  20. In vivo inhibition of acylpeptide hydrolase by carbapenem antibiotics causes the decrease of plasma concentration of valproic acid in dogs.

    PubMed

    Suzuki, Eiko; Nakai, Daisuke; Ikenaga, Hidenori; Fusegawa, Keiichi; Goda, Ryoya; Kobayashi, Nobuhiro; Kuga, Hiroshi; Izumi, Takashi

    2016-01-01

    1. Our previous in vitro studies suggest that inhibition of the acylpeptide hydrolase (APEH) activity as valproic acid glucuronide (VPA-G) hydrolase by carbapenems in human liver cytosol is a key process for clinical drug-drug interaction (DDI) of valproic acid (VPA) with carbapenems. Here, we investigated whether in vivo DDI of VPA with meropenem (MEPM) was caused via inhibition of APEH in dogs. 2. More rapid decrease of plasma VPA levels and increased urinary excretion of VPA-G were observed after co-administration with MEPM compared with those after without co-administration, whereas the plasma level and bile excretion of VPA-G showed no change. 3. Dog VPA-G hydrolase activity, inhibited by carbapenems, was mainly located in cytosol from both the liver and kidney. APEH-immunodepleted cytosols lacked VPA-G hydrolase activity. Hepatic and renal APEH activity was negligible even at 24 h after dosing of MEPM to a dog. 4. In conclusion, DDI of VPA with carbapenems in dogs is caused by long-lasting inhibition of APEH-mediated VPA-G hydrolysis by carbapenems, which could explain the delayed recovery of plasma VPA levels to the therapeutic window even after discontinuation of carbapenems in humans.

  1. Inhibition of fatty acid amide hydrolase by kaempferol and related naturally occurring flavonoids

    PubMed Central

    Thors, L; Belghiti, M; Fowler, C J

    2008-01-01

    Background and purpose: Recent studies have demonstrated that the naturally occurring isoflavone compounds genistein and daidzein inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) in the low micromolar concentration range. The purpose of the present study was to determine whether this property is shared by flavonoids. Experimental approach: The hydrolysis of anandamide in homogenates and intact cells was measured using the substrate labelled in the ethanolamine part of the molecule. Key results: Twenty compounds were tested. Among the commonly occurring flavonoids, kaempferol was the most potent, inhibiting FAAH in a competitive manner with a Ki value of 5 μM. Among flavonoids with a more restricted distribution in nature, the two most active toward FAAH were 7-hydroxyflavone (IC50 value of 0.5–1 μM depending on the solvent used) and 3,7-dihydroxyflavone (IC50 value 2.2 μM). All three compounds reduced the FAAH-dependent uptake of anandamide and its metabolism by intact RBL2H3 basophilic leukaemia cells. Conclusions and implications: Inhibition of FAAH is an additional in vitro biochemical property of flavonoids. Kaempferol, 7-hydroxyflavone and 3,7-dihydroxyflavone may be useful as templates for the synthesis of novel compounds, which target several systems that are involved in the control of inflammation and cancer. PMID:18552875

  2. The role of fatty acid amide hydrolase inhibition in nicotine reward and dependence

    PubMed Central

    Muldoon, Pretal P.; Lichtman, Aron H.; Parsons, Loren H.; Damaj, M. Imad

    2012-01-01

    The endogenous cannabinoid anandamide (AEA) exerts the majority of its effects at CB1 and CB2 receptors and is degraded by fatty acid amide hydrolase (FAAH). FAAH KO mice and animals treated with FAAH inhibitors are impaired in their ability to hydrolyze AEA and other non-cannabinoid lipid signaling molecules, such as oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). AEA and these other substrates activate non- cannabinoid receptor systems, including TRPV1 and PPAR-α receptors. In this mini review, we describe the functional consequences of FAAH inhibition on nicotine reward and dependence as well as the underlying endocannabinoid and non-cannabinoid receptor systems mediating these effects. Interestingly, FAAH inhibition seems to mediate nicotine dependence differently in mice and rats. Indeed, pharmacological and genetic FAAH disruption in mice enhances nicotine reward and withdrawal. However, in rats, pharmacological blockade of FAAH significantly inhibits nicotine reward and has no effect in nicotine withdrawal. Studies suggest that non-cannabinoid mechanisms may play a role in these species differences. PMID:22705310

  3. Colorimetric assay for S-adenosylhomocysteine hydrolase activity and inhibition using fluorosurfactant-capped gold nanoparticles.

    PubMed

    Lin, Jia-Hui; Chang, Chung-Wei; Wu, Zong-Han; Tseng, Wei-Lung

    2010-11-01

    This study reports a simple colorimetric method for the sensitive detection of S-adenosylhomocysteine hydrolase (SAHH) activity and inhibition using fluorosurfactant-capped gold nanoparticles (FSN-AuNPs). FSN stabilizes the AuNPs against conditions of high ionic strength, and FSN-AuNPs are merely aggregated in the presence of homocysteine (HCys) and cysteine. Because of this feature, FSN-AuNPs were found to be dispersed in the presence of S-adenosylhomocysteine (SAH) that lacks a free thiol group. After SAHH catalyzed the hydrolysis of SAH, the produced HCys molecules were bound to the surface of AuNPs through the formation of Au-S bonds. As a result, the nanoparticle (NP) aggregation occurred through electrostatic attraction between each HCys-attached AuNP. This approach had a minimum detectable concentration of 100 units/L (~6 nM). Additionally, because adenosine analogs are capable of inhibiting SAHH activity, the addition of adenosine analogs to a solution containing SAH and SAHH resulted in the suppression of hydrolyzed SAH-induced NP aggregation. Adenosine analogs exhibited the following trend in the half-maximal inhibitory concentrations: adenosine > adenosine monophosphate > adenosine diphosphate ~ adenosine triphosphate. We have demonstrated that the combination of SAHH inhibition and FSN-AuNPs can be utilized for the selective detection of adenosine.

  4. The ω-3 epoxide of eicosapentaenoic acid inhibits endothelial cell proliferation by p38 MAP kinase activation and cyclin D1/CDK4 down-regulation

    PubMed Central

    Cui, Pei H; Petrovic, Nenad; Murray, Michael

    2011-01-01

    BACKGROUND AND PURPOSE Dietary intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) like eicosapentaenoic acid (EPA) decreases cancer risk, while arachidonic acid and other ω-6 PUFAs increase risk, but the underlying mechanisms are unclear. Cytochrome P450 (CYP)-derived epoxides contribute to enhanced tumourigenesis due to ω-6 PUFA intake. Thus, ω-6 arachidonic acid epoxides (EETs) inhibit apoptosis and stimulate proliferation by up-regulating cyclin D1 expression in cells. The present study evaluated the corresponding ω-3 PUFA epoxides and assessed their role in the regulation of cell proliferation. EXPERIMENTAL APPROACH Four chemically stable EPA epoxides (formed at the 8,9-, 11,12-, 14,15- and 17,18-olefinic bonds) were synthesized and tested against growth-related signalling pathways in brain microvascular endothelial bEND.3 cells. Cell cycle distribution was determined by flow cytometry and cyclin gene expression by immunoblotting and real-time PCR. The role of the p38 mitogen-activated protein (MAP) kinase in cyclin D1 dysregulation was assessed using specific inhibitors and dominant-negative expression plasmids. KEY RESULTS The ω-3 17,18-epoxide of EPA decreased cell proliferation, interrupted the cell cycle in S-phase and down-regulated the cyclin D1/cyclin-dependent kinase (CDK)-4 complex, whereas the 8,9-, 11,12- and 14,15-epoxides were either inactive or enhanced proliferation. Cyclin D1 down-regulation by 17,18-epoxy-EPA was mediated by activation of the growth-suppressing p38 MAP kinase, but the alternate EPA-epoxides were inactive. CONCLUSIONS AND IMPLICATIONS The present findings suggest that the epoxide formed by CYP enzymes at the ω-3 olefinic bond may contribute to the beneficial effects of ω-3 PUFA by down-regulating cyclin D1 and suppressing cell proliferation. PMID:21077851

  5. Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition.

    PubMed

    Chang, Jae Won; Cognetta, Armand B; Niphakis, Micah J; Cravatt, Benjamin F

    2013-07-19

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo, using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and α-β hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome.

  6. Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition

    PubMed Central

    Chang, Jae Won; Cognetta, Armand B.; Niphakis, Micah J.; Cravatt, Benjamin F.

    2013-01-01

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and alpha-beta hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome. PMID:23701408

  7. Inhibition of xenobiotic-degrading hydrolases by organophosphinates. Annual progress report No. 1 Jul 82-1 Jul 83

    SciTech Connect

    Brown, T.M.; Zimmerman, J.K.; Bryson, P.K.; Grothusen, J.R.

    1983-07-01

    Organophosphinate pretreatment agents for chemical warfare defense inhibited carboxylester hydrolase from porcine liver and from rabbit liver. Recovery of rabbit liver monomeric carboxylester hydrolase to at least 30% of its initial activity was observed 48 hr. after inhibition by certain 4-nitrophenyl alkyl(phenyl)phosphinates and analogues. When ranked according to the initial rates at which their phosphinylated enzymes recovered, they were methyl(phenyl)>methyl(2-thienyl)>methyl(2-furyl)>ethyl(phenyl)>di-2-thienyl>diphenyl. Recovery was less than 20% in 96 hr. following inhibition by methyl(naphthyl),di-2-furyl, isopropyl(phenyl), dichloromethyl(phenyl), and bis chloromethyl substituted analogues. High performance liquid chromatography on silica using 10% to 20% 2-propanol in hexane as mobile phase resulted in satisfactory chromatograms for all except the most polar phosphinates. This method was useful in determining purity and decomposition of the compounds. Arylester hydrolase was purified 30-fold from rabbit serum by a sequence of polyethylene glycol fractionation, ion exchange chromatography, ammonium sulfate fractionation, molecular exclusion chromatography and pseudo-affinity chromatography. The partially purified enzyme was activated by 2.5 mM divalent calcium.

  8. A2E-epoxides damage DNA in retinal pigment epithelial cells. Vitamin E and other antioxidants inhibit A2E-epoxide formation.

    PubMed

    Sparrow, Janet R; Vollmer-Snarr, Heidi R; Zhou, Jilin; Jang, Young P; Jockusch, Steffen; Itagaki, Yasuhiro; Nakanishi, Koji

    2003-05-16

    The autofluorescent pigments that accumulate in retinal pigment epithelial cells with aging and in some retinal disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum being most damaging. Efforts to understand the events precipitating the death of the cells have revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds. Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit reactivity toward DNA with oxidative base changes being at least one of these lesions. Mass spectrometry revealed that the antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis and cell viability studies revealed a corresponding reduction in the incidence of DNA damage and cell death. Vitamin E, a lipophilic antioxidant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both vitamins simultaneously did not confer additional benefit. Studies in which singlet oxygen was generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.

  9. Excessive ATP hydrolysis in ischemic myocardium by mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity.

    PubMed

    Grover, Gary J; Atwal, Karnail S; Sleph, Paul G; Wang, Feng-Li; Monshizadegan, Hossain; Monticello, Thomas; Green, David W

    2004-10-01

    Mitochondrial F(1)F(0)-ATPase normally synthesizes ATP in the heart, but under ischemic conditions this enzyme paradoxically causes ATP hydrolysis. Nonselective inhibitors of this enzyme (aurovertin, oligomycin) inhibit ATP synthesis in normal tissue but also inhibit ATP hydrolysis in ischemic myocardium. We characterized the profile of aurovertin and oligomycin in ischemic and nonischemic rat myocardium and compared this with the profile of BMS-199264, which only inhibits F(1)F(0)-ATP hydrolase activity. In isolated rat hearts, aurovertin (1-10 microM) and oligomycin (10 microM), at concentrations inhibiting ATPase activity, reduced ATP concentration and contractile function in the nonischemic heart but significantly reduced the rate of ATP depletion during ischemia. They also inhibited recovery of reperfusion ATP and contractile function, consistent with nonselective F(1)F(0)-ATPase inhibitory activity, which suggests that upon reperfusion, the hydrolase activity switches back to ATP synthesis. BMS-199264 inhibits F(1)F(0) hydrolase activity in submitochondrial particles with no effect on ATP synthase activity. BMS-199264 (1-10 microM) conserved ATP in rat hearts during ischemia while having no effect on preischemic contractile function or ATP concentration. Reperfusion ATP levels were replenished faster and necrosis was reduced by BMS-199264. ATP hydrolase activity ex vivo was selectively inhibited by BMS-199264. Therefore, excessive ATP hydrolysis by F(1)F(0)-ATPase contributes to the decline in cardiac energy reserve during ischemia and selective inhibition of ATP hydrolase activity can protect ischemic myocardium.

  10. Tissue and stage-specific juvenile hormone esterase (JHE) and epoxide hydrolase (JHEH) enzyme activities and Jhe transcript abundance in lines of the cricket Gryllus assimilis artificially selected for plasma JHE activity: implications for JHE microevolution.

    PubMed

    Anand, Anurag; Crone, Erica J; Zera, Anthony J

    2008-09-01

    Fat body and midgut juvenile hormone esterase (JHE) and juvenile hormone epoxide hydrolase (JHEH) specific activities, and plasma JHE activity, were measured throughout the last stadium in two pairs (blocks) of lines of the cricket Gryllus assimilis, each pair of which had been artificially selected for high- or low-plasma JHE activity. Highly significant differences were observed between high- and low-activity lines of each block on most days for fat body JHE, and on one day for midgut JHE activity. In each block, line differences in developmental profiles for fat body JHE activity paralleled line differences in plasma JHE activity during the early-mid stadium, but not during the latter part of the stadium. The developmental profile of midgut JHE activity differed from that of plasma and fat body JHE activity, exhibiting peaks during the early and latter parts of the stadium. Midgut and fat body JHEH activities exhibited a mid-stadium peak in all lines, but activities were very similar in all lines. Fat body JHE appears to be a more significant contributor to plasma JHE than is midgut JHE. During the middle of the last stadium (day 4), Jhe transcript abundance was significantly higher in fat body or midgut of high- vs. low-JHE-activity lines. Jhe transcript abundance was positively correlated with JHE enzyme activity in either fat body or midgut, and with plasma JHE activity. Natural populations of G. assimilis harbor genetic variation for Jhe gene expression which appears to contribute to genetic variation in JHE specific activity in fat body and midgut. These genes appear to have been the targets of artificial selection that resulted in lines that differ dramatically in high- or low-plasma JHE activity. These genes appear to have little, if any, pleiotropic effects on JHEH specific activity.

  11. Effect of inhibition of fatty acid amide hydrolase on MPTP-induced dopaminergic neuronal damage.

    PubMed

    Viveros-Paredes, J M; Gonzalez-Castañeda, R E; Escalante-Castañeda, A; Tejeda-Martínez, A R; Castañeda-Achutiguí, F; Flores-Soto, M E

    2017-01-16

    Parkinson's disease (PD) is a neurodegenerative disorder characterised by balance problems, muscle rigidity, and slow movement due to low dopamine levels and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The endocannabinoid system is known to modulate the nigrostriatal pathway through endogenous ligands such as anandamide (AEA), which is hydrolysed by fatty acid amide hydrolase (FAAH). The purpose of this study was to increase AEA levels using FAAH inhibitor URB597 to evaluate the modulatory effect of AEA on dopaminergic neuronal death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our study included 4 experimental groups (n = 6 mice per group): a control group receiving no treatment, a group receiving URB597 (0.2mg/kg) every 3 days for 30 days, a group treated with MPTP (30mg/kg) for 5 days, and a group receiving URB597 and subsequently MPTP injections. Three days after the last dose, we conducted a series of behavioural tests (beam test, pole test, and stride length test) to compare motor coordination between groups. We subsequently analysed immunoreactivity of dopaminergic cells and microglia in the SNpc and striatum. Mice treated with URB597 plus MPTP were found to perform better on behavioural tests than mice receiving MPTP only. According to the immunohistochemistry study, mice receiving MPTP showed fewer dopaminergic cells and fibres in the SNpc and striatum. Animals treated with URB597 plus MPTP displayed increased tyrosine hydroxylase immunoreactivity compared to those treated with MPTP only. Regarding microglial immunoreactivity, the group receiving MPTP showed higher Iba1 immunoreactivity in the striatum and SNpc than did the group treated with URB597 plus MPTP. Our results show that URB597 exerts a protective effect since it inhibits dopaminergic neuronal death, decreases microglial immunoreactivity, and improves MPTP-induced motor alterations. Copyright © 2016 Sociedad Española de Neurología. Publicado

  12. Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate*

    PubMed Central

    Boyd, Jeffrey M.; Clark, Daniel D.; Kofoed, Melissa A.; Ensign, Scott A.

    2010-01-01

    The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys82) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys87 was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys82. These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis. PMID:20551308

  13. Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions.

    PubMed

    Stiborová, Marie; Moserová, Michaela; Černá, Věra; Indra, Radek; Dračínský, Martin; Šulc, Miroslav; Henderson, Colin J; Wolf, C Roland; Schmeiser, Heinz H; Phillips, David H; Frei, Eva; Arlt, Volker M

    2014-04-06

    In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b5, and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼ 1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.

  14. Lipid Sulfates and Sulfonates Are Allosteric Competitive Inhibitors of the N-Terminal Phosphatase Activity of the Mammalian Soluble Epoxide Hydrolase†

    PubMed Central

    Tran, Katherine L.; Aronov, Pavel A.; Tanaka, Hiromasa; Newman, John W.; Hammock, Bruce D.; Morisseau, Christophe

    2006-01-01

    The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with KI in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH. PMID:16142916

  15. Effect of alpha lipoic acid on leukotriene A4 hydrolase.

    PubMed

    Torres, María José; Fierro, Angélica; Pessoa-Mahana, C David; Romero-Parra, Javier; Cabrera, Gonzalo; Faúndez, Mario

    2017-03-15

    Leukotriene A4 hydrolase is a soluble enzyme with epoxide hydrolase and aminopeptidase activities catalysing the conversion of leukotriene A4 to leukotriene B4 and the hydrolysis of the peptide proline-glycine-proline. Imbalances in leukotriene B4 synthesis are related to several pathologic conditions. Currently there are no available drugs capable to modulate the synthesis of leukotriene B4 or to block its receptors. Here we show the inhibitory profile of alpha lipoic acid on the activity of leukotriene A4 Hydrolase. Alpha lipoic acid inhibited both activities of the enzyme at concentrations lower than 10μM. The 5-lipoxygenase inhibitor zileuton, or the 5-lipoxygenase activating protein inhibitor MK-886, were unable to inhibit the activity of the enzyme. Acute promyelocytic leukaemia HL-60 cells were differentiated to leukotriene A4 hydrolase expressing neutrophil-like cells. Alpha lipoic acid inhibited the aminopeptidase activity of the cytosolic fraction from neutrophil-like cells but had no effect on the cytosolic fraction from undifferentiated cells. Docking and molecular dynamic approximations revealed that alpha lipoic acid participates in electrostatic interactions with K-565 and R-563, which are key residues for the carboxylate group recognition of endogenous substrates by the enzyme. Alpha lipoic acid is a compound widely used in clinical practice, most of its therapeutic effects are associated with its antioxidants properties, however, antioxidant effect alone is unable to explain all clinical effects observed with alpha lipoic acid. Our results invite to evaluate the significance of the inhibitory effect of alpha lipoic acid on the catalytic activity of leukotriene A4 hydrolase using in vivo models.

  16. Heptachlor epoxide

    Integrated Risk Information System (IRIS)

    Heptachlor epoxide ; CASRN 1024 - 57 - 3 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogen

  17. Delta12-Prostaglandin J2 inhibits the ubiquitin hydrolase UCH-L1 and elicits ubiquitin-protein aggregation without proteasome inhibition.

    PubMed

    Li, Zongmin; Melandri, Francesco; Berdo, Ingrid; Jansen, Marlon; Hunter, Lavonne; Wright, Saundrene; Valbrun, Danielle; Figueiredo-Pereira, Maria E

    2004-07-09

    To investigate molecular mechanisms linking inflammation with neurodegeneration, we treated neuronal cultures with prostaglandins (PGs), which are mediators of inflammation. PGA1, D2, J2, and Delta12-PGJ2, but not PGE2, reduced the viability and raised the levels of ubiquitinated proteins in the neuronal cells. PGJ2 and its metabolite, Delta12-PGJ2, were the most potent of the four neurotoxic PGs tested in inducing both effects. To address the mechanism by which these agents lead to the accumulation of ubiquitinated proteins, we tested their effects on neuronal ubiquitin hydrolases UCH-L1 and UCH-L3 as well as on proteasome activity. Notably, Delta12-PGJ2 inhibited the activities of UCH-L1 (K(i) approximately 3.5 microM) and UCH-L3 (K(i) approximately 8.1 microM) without affecting proteasome activity. Intracellular aggregates containing ubiquitinated proteins were detected in Delta12-PGJ2-treated cells, indicating that these aggregates can form independently of proteasome inhibition. In conclusion, impairment of ubiquitin hydrolase activity, such as triggered by Delta12-PGJ2, may be an important contributor to neurodegeneration associated with accumulation of ubiquitinated proteins and inflammation.

  18. Cellular viability effects of fatty acid amide hydrolase inhibition on cerebellar neurons

    PubMed Central

    2011-01-01

    The endocannabinoid anandamide (ANA) participates in the control of cell death inducing the formation of apoptotic bodies and DNA fragmentation. The aim of this study was to evaluate whether the ANA degrading enzyme, the fatty acid amide hydrolase (FAAH), would induce cellular death. Experiments were performed in cerebellar granule neurons cultured with the FAAH inhibitor, URB597 (25, 50 or 100 nM) as well as endogenous lipids such as oleoylethanolamide (OEA) or palmitoylethanolamide (PEA) and cellular viability was determined by MTT test. Neurons cultured with URB597 (25, 50 or 100 nM) displayed a decrease in cellular viability. In addition, if cultured with OEA (25 nM) or PEA (100 nM), cellular death was found. These results further suggest that URB597, OEA or PEA promote cellular death. PMID:21854612

  19. Identification of benzofuran central cores for the inhibition of leukotriene A(4) hydrolase.

    PubMed

    Eccles, Wendy; Blevitt, Jonathan M; Booker, Jamila N; Chrovian, Christa C; Crawford, Shelby; de Leon, Aimee Rose; Deng, Xiaohu; Fourie, Anne M; Grice, Cheryl A; Herman, Krystal; Karlsson, Lars; Kearney, Aaron M; Lee-Dutra, Alice; Liang, Jimmy; Luna, Rosa; Pippel, Dan; Rao, Navin; Riley, Jason P; Santillán, Alejandro; Savall, Brad; Tanis, Virginia M; Xue, Xiaohua; Young, Arlene L

    2013-02-01

    Leukotrienes (LT's) are known to play a physiological role in inflammatory immune response. Leukotriene A(4) hydrolase (LTA(4)H) is a cystolic enzyme that stereospecifically catalyzes the transformation of LTA(4) to LTB(4). LTB(4) is a known pro-inflammatory mediator. This paper describes the identification and synthesis of substituted benzofurans as LTH(4)H inhibitors. The benzofuran series demonstrated reduced mouse and human whole blood LTB(4) levels in vitro and led to the identification one analog for advanced profiling. Benzofuran 28 showed dose responsive target engagement and provides a useful tool to explore a LTA(4)H inhibitor for the treatment of inflammatory diseases, such as asthma and inflammatory bowel disease (IBD).

  20. Long-term consequences of perinatal fatty acid amino hydrolase inhibition

    PubMed Central

    Wu, Chia-Shan; Morgan, Daniel; Jew, Chris P; Haskins, Chris; Andrews, Mary-Jeanette; Leishman, Emma; Spencer, Corinne M; Czyzyk, Traci; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2014-01-01

    Background and PurposeFatty acid amide hydrolase inhibitors show promise as a treatment for anxiety, depression and pain. Here we investigated whether perinatal exposure to URB597, a fatty acid amide hydrolase inhibitor, alters brain development and affects behaviour in adult mice. Experimental ApproachMouse dams were treated daily from gestational day 10.5 to 16.5 with 1, 3 or 10 mg kg−1 URB597. MS was used to measure a panel of endocannabinoids and related lipid compounds and brain development was assessed at embryonic day 16.5. Separate cohorts of mouse dams were treated with 10 mg kg−1 URB597, from gestational day 10.5 to postnatal day 7, and the adult offspring were assessed with a battery of behavioural tests. Key ResultsPerinatal URB597 exposure elevated anandamide and related N-acyl amides. URB597 did not induce signs of toxicity or affect dam weight gain, neurogenesis or axonal development at embryonic day 16.5. It did lead to subtle behavioural deficits in adult offspring, manifested by reduced cocaine-conditioned preference, increased depressive behaviours and impaired working memory. Anxiety levels, motor function and sensory-motor gating were not significantly altered. Conclusions and ImplicationsTaken together, the present results highlight how exposure to elevated levels of anandamide and related N-acyl amides during brain development can lead to subtle alterations in behaviour in adulthood. Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6 PMID:24730060

  1. Long-term consequences of perinatal fatty acid amino hydrolase inhibition.

    PubMed

    Wu, Chia-Shan; Morgan, Daniel; Jew, Chris P; Haskins, Chris; Andrews, Mary-Jeanette; Leishman, Emma; Spencer, Corinne M; Czyzyk, Traci; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2014-03-01

    Fatty acid amide hydrolase inhibitors show promise as a treatment for anxiety, depression and pain. Here we investigated whether perinatal exposure to URB597, a fatty acid amide hydrolase inhibitor, alters brain development and affects behaviour in adult mice. Mouse dams were treated daily from gestational day 10.5 to 16.5 with 1, 3 or 10 mg kg−1 URB597. MS was used to measure a panel of endocannabinoids and related lipid compounds and brain development was assessed at embryonic day 16.5. Separate cohorts of mouse dams were treated with 10 mg kg−1 URB597, from gestational day 10.5 to postnatal day 7, and the adult offspring were assessed with a battery of behavioural tests. Perinatal URB597 exposure elevated anandamide and related N-acyl amides. URB597 did not induce signs of toxicity or affect dam weight gain, neurogenesis or axonal development at embryonic day 16.5. It did lead to subtle behavioural deficits in adult offspring, manifested by reduced cocaine-conditioned preference, increased depressive behaviours and impaired working memory. Anxiety levels, motor function and sensory-motor gating were not significantly altered. Taken together, the present results highlight how exposure to elevated levels of anandamide and related N-acyl amides during brain development can lead to subtle alterations in behaviour in adulthood. This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6

  2. Biotechnological production of enantiopure epoxides by enzymatic kinetic resolution.

    PubMed

    Choi, Won Jae

    2009-08-01

    Enantiopure epoxides are high value-added synthons for the production of pharmaceuticals, agrochemicals, as well as versatile fine chemicals and have broad scope of market demand for their applications. A major challenge in conventional organic synthesis is to generate such compounds in high enantiopurity with reasonable yield. Among possible chemical and biological technologies for enantiopure epoxide preparation, enzymatic kinetic resolution has been paid much attention with respect to its high enantioselectivity. Epoxide hydrolase (EH) has shown promising characteristics for the preparation of enantiopure epoxides and vicinal diols during enantioselective hydrolysis of racemic epoxides. EH is readily available from microbial resources thus it is being employed for biohydrolysis of a variety of epoxides. Recent technical progress in EH-catalyzed enantioselective hydrolysis is summarized in terms of exploration of novel EH, its functional improvement, high throughput assay, and preparative scale resolution process.

  3. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening

    PubMed Central

    Bachovchin, Daniel A.; Ji, Tianyang; Li, Weiwei; Simon, Gabriel M.; Blankman, Jacqueline L.; Adibekian, Alexander; Hoover, Heather; Niessen, Sherry; Cravatt, Benjamin F.

    2010-01-01

    Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo. PMID:21084632

  4. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening.

    PubMed

    Bachovchin, Daniel A; Ji, Tianyang; Li, Weiwei; Simon, Gabriel M; Blankman, Jacqueline L; Adibekian, Alexander; Hoover, Heather; Niessen, Sherry; Cravatt, Benjamin F

    2010-12-07

    Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.

  5. Parabens inhibit fatty acid amide hydrolase: A potential role in paraben-enhanced 3T3-L1 adipocyte differentiation.

    PubMed

    Kodani, Sean D; Overby, Haley B; Morisseau, Christophe; Chen, Jiangang; Zhao, Ling; Hammock, Bruce D

    2016-11-16

    Parabens are a class of small molecules that are regularly used as preservatives in a variety of personal care products. Several parabens, including butylparaben and benzylparaben, have been found to interfere with endocrine signaling and to stimulate adipocyte differentiation. We hypothesized these biological effects could be due to interference with the endocannabinoid system and identified fatty acid amide hydrolase (FAAH) as the direct molecular target of parabens. FAAH inhibition by parabens yields mixed-type and time-independent kinetics. Additionally, structure activity relationships indicate FAAH inhibition is selective for the paraben class of compounds and the more hydrophobic parabens have higher potency. Parabens enhanced 3T3-L1 adipocyte differentiation in a dose dependent fashion, different from two other FAAH inhibitors URB597 and PF622. Moreover, parabens, URB597 and PF622 all failed to enhance AEA-induced differentiation. Furthermore, rimonabant, a cannabinoid receptor 1 (CB1)-selective antagonist, did not attenuate paraben-induced adipocyte differentiation. Thus, adipogenesis mediated by parabens likely occurs through modulation of endocannabinoids, but cell differentiation is independent of direct activation of CB1 by endocannabinoids. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities.

    PubMed

    Castro, D P; Figueiredo, M B; Genta, F A; Ribeiro, I M; Tomassini, T C B; Azambuja, P; Garcia, E S

    2009-06-01

    The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A(2) (PLA(2)) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1mug/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10microg/insect) or PAF (1microg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA(2) activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.

  7. Cholesterol-5,6-epoxides: chemistry, biochemistry, metabolic fate and cancer.

    PubMed

    Poirot, Marc; Silvente-Poirot, Sandrine

    2013-03-01

    In the nineteen sixties it was proposed that cholesterol might be involved in the etiology of cancers and cholesterol oxidation products were suspected of being causative agents. Researchers had focused their attention on cholesterol-5,6-epoxides (5,6-ECs) based on several lines of evidence: 1) 5,6-ECs contained an oxirane group that was supposed to confer alkylating properties such as those observed for aliphatic and aromatic epoxides. 2) cholesterol-5,6-epoxide hydrolase (ChEH) was induced in pre-neoplastic lesions of skin from rats exposed to ultraviolet irradiations and ChEH was proposed to be involved in detoxification processes like other epoxide hydrolases. However, 5,6-ECs failed to induce carcinogenicity in rodents which ruled out a potent carcinogenic potential for 5,6-ECs. Meanwhile, clinical studies revealed an anomalous increase in the concentrations of 5,6β-EC in the nipple fluids of patients with pre-neoplastic breast lesions and in the blood of patients with endometrious cancers, suggesting that 5,6-ECs metabolism could be linked with cancer. Paradoxically, ChEH has been recently shown to be totally inhibited by therapeutic concentrations of tamoxifen (Tam), which is one of the main drugs used in the hormonotherapy and the chemoprevention of breast cancers. These data would suggest that the accumulation of 5,6-ECs could represent a risk factor, but we found that 5,6-ECs were involved in the induction of breast cancer cell differentiation and death induced by Tam suggesting a positive role of 5,6-ECs. These observations meant that the biochemistry and the metabolism of 5,6-ECs needed to be extensively studied. We will review the current knowledge and the future direction of 5,6-ECs chemistry, biochemistry, metabolism, and relationship with cancer.

  8. Inhibition of fatty acid amide hydrolase (FAAH) reduces spinal nociceptive responses and expression of spinal long-term potentiation (LTP).

    PubMed

    Eriksen, Guro S; Jacobsen, Line Melå; Mahmood, Aqsa; Pedersen, Linda M; Gjerstad, Johannes

    2012-02-10

    Fatty acid amide hydrolase (FAAH) is an enzyme that metabolizes endocannabinoids and fatty acid amides possibly linked to activation of the opioid system. To examine how this enzyme affects spinal signalling, electrophysiological recordings in the dorsal horn and qPCR on dorsal horn tissue following systemic administration of the FAAH inhibitor URB597 (0.3 and 1.0mg/kg i.v.) and spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.), were performed. The present data showed that the suppressive effect of the FAAH inhibitor URB597 (1.0mg/kg i.v.) on the spinal nociceptive responses was prevented by spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.). Moreover, the present findings demonstrated that the FAAH inhibitor URB597 (1.0mg/kg i.v.) partly reversed expression of spinal long-term potentiation (LTP) and also attenuated the LTP-associated increased Zif expression. We conclude that pharmacological inactivation of FAAH may be a promising strategy to inhibit the development of central hyperalgesia; thereby reinforcing the role of FAAH as a potential therapeutic target.

  9. Full Fatty Acid Amide Hydrolase Inhibition Combined with Partial Monoacylglycerol Lipase Inhibition: Augmented and Sustained Antinociceptive Effects with Reduced Cannabimimetic Side Effects in Mice

    PubMed Central

    Ghosh, Sudeshna; Kinsey, Steven G.; Liu, Qing-song; Hruba, Lenka; McMahon, Lance R.; Grim, Travis W.; Merritt, Christina R.; Wise, Laura E.; Abdullah, Rehab A.; Selley, Dana E.; Sim-Selley, Laura J.; Cravatt, Benjamin F.

    2015-01-01

    Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the primary hydrolytic enzymes for the respective endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonylglycerol (2-AG), produces antinociception but with minimal cannabimimetic side effects. Although selective inhibitors of either enzyme often show partial efficacy in various nociceptive models, their combined blockade elicits augmented antinociceptive effects, but side effects emerge. Moreover, complete and prolonged MAGL blockade leads to cannabinoid receptor type 1 (CB1) receptor functional tolerance, which represents another challenge in this potential therapeutic strategy. Therefore, the present study tested whether full FAAH inhibition combined with partial MAGL inhibition would produce sustained antinociceptive effects with minimal cannabimimetic side effects. Accordingly, we tested a high dose of the FAAH inhibitor PF-3845 (N-​3-​pyridinyl-​4-​[[3-​[[5-​(trifluoromethyl)-​2-​pyridinyl]oxy]phenyl]methyl]-​1-​piperidinecarboxamide; 10 mg/kg) given in combination with a low dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate] (4 mg/kg) in mouse models of inflammatory and neuropathic pain. This combination of inhibitors elicited profound increases in brain AEA levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels. This combination produced significantly greater antinociceptive effects than single enzyme inhibition and did not elicit common cannabimimetic effects (e.g., catalepsy, hypomotility, hypothermia, and substitution for Δ9-tetrahydrocannabinol in the drug-discrimination assay), although these side effects emerged with high-dose JZL184 (i.e., 100 mg/kg). Finally, repeated administration of this combination did not lead to tolerance to its antiallodynic actions in the carrageenan assay or CB1 receptor functional tolerance. Thus, full FAAH inhibition combined

  10. Concurrent Cooperativity and Substrate Inhibition in the Epoxidation of Carbamazepine by Cytochrome P450 3A4 Active Site Mutants Inspired by Molecular Dynamics Simulations

    PubMed Central

    2015-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V. PMID:25545162

  11. Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.

    PubMed

    Müller, Christian S; Knehans, Tim; Davydov, Dmitri R; Bounds, Patricia L; von Mandach, Ursula; Halpert, James R; Caflisch, Amedeo; Koppenol, Willem H

    2015-01-27

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.

  12. Cephalosporin-induced alteration in hepatic glutathione redox state. A potential mechanism for inhibition of hepatic reduction of vitamin K1,2,3-epoxide in the rat.

    PubMed Central

    Mitchell, M C; Mallat, A; Lipsky, J J

    1990-01-01

    Hypoprothrombinemia is a serious adverse effect of antimicrobial therapy that occurs after administration of some second- and third-generation cephalosporins which contain the methyltetrazole-thiol (MTT) group. Previous studies have shown that in vitro MTT directly inhibits microsomal gamma-carboxylation of a synthetic pentapeptide. Since MTT is a thiocarbamide, a type of compound that can increase oxidation of glutathione, the present studies were carried out to determine whether alterations in hepatic glutathione redox state might interfere with vitamin K metabolism. Dose-related increases in biliary efflux and hepatic concentration of oxidized glutathione (GSSG) occurred after intravenous administration of MTT or MTT-containing antibiotics to rats. This finding suggested that these compounds could alter the hepatic glutathione redox state in vivo. Microsomal reduction of vitamin K epoxide occurred in the presence of 100 microM dithiothreitol (DTT), but was inhibited by preincubation with GSSG at concentrations as low as 10 microM. At higher concentrations of DTT (1.0 mM) inhibition by GSSG persisted, but higher concentrations were required, suggesting that the thiol/disulfide ratio, rather than the absolute concentration of GSSG was important. By contrast, GSSG did not effect microsomal gamma-carboxylation of a pentapeptide, using either vitamin K1 or its hydroquinone as a cofactor. These findings suggest a novel mechanism for the hypoprothrombinemia occurring after administration of MTT-containing antibiotics. PMID:1978724

  13. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    ERIC Educational Resources Information Center

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  14. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    ERIC Educational Resources Information Center

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  15. Purification and characterization of porcine skeletal muscle aminopeptidase T, a novel metallopeptidase homologous to leukotriene A4 hydrolase.

    PubMed

    Sarker, Mohammed Alamgir; Matsuda, Shinji; Mizutani, Osamu; Rao, Shengbin; Migita, Koshiro; Goto-Yamamoto, Nami; Iefuji, Haruyuki; Nishimura, Toshihide

    2011-01-01

    A novel aminopeptidase, Aminopeptidase T (APase T), was purified from porcine skeletal muscle following successive column chromatography: twice on DEAE-cellulose, hydroxyapatite, and Sephacryl S-200 HR using Leu-β-naphthylamide (LeuNap) as a substrate. The molecular mass of the enzyme was 69 kDa on SDS-PAGE. The optimum pH towards LeuNap of the enzyme was about 7. The enzyme activity was strongly inhibited by bestatin and was negatively affected by ethylenediaminetetraacetic acid (EDTA). Chlorine-activated APase T liberated Leu, Ala, Met, Pro, and Arg from Nap derivatives. The APase T gene consisted of an ORF of 1,836 bp encoding a protein of 611 amino acid residues. The APase T was highly homologous to bovine, human, and mouse Leukotriene A(4) hydrolase (LTA(4)H), a bifunctional enzyme which exhibits APase and epoxide hydrolase activity.

  16. Effect of epoxides and α-methylene-γ-lactone skeleton of sesquiterpenes from yacon (Smallanthus sonchifolius) leaves on caspase-dependent apoptosis and NF-κB inhibition in human cercival cancer cells.

    PubMed

    Siriwan, Dalad; Naruse, Takayuki; Tamura, Hirotoshi

    2011-10-01

    The present study investigated the cytotoxicity of enhydrin (1), uvedalin (2) and sonchifolin (3) in cervical cancer cells. We have found that SLs 1-3 in doses in range of 0.22-10 μM inhibited cell proliferation and induced apoptosis in both a dose- and time-dependent fashion. A significant cell death induction was supported by morphological studies. The apoptotic effect is associated with caspase-3/7 activation and NF-кB inhibition. Interestingly, enhydrin possessing two epoxide units was found to be the most cytotoxic compound. Therefore it can be assumed that number of epoxides and existence of α-methylene-γ-lactone moiety are essential for the acceleration of apoptosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.

    PubMed

    Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

    2006-01-01

    Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway.

  18. Low-dose benzo(a)pyrene and its epoxide metabolite inhibit myogenic differentiation in human skeletal muscle-derived progenitor cells.

    PubMed

    Chiu, Chen-Yuan; Yen, Yuan-Peng; Tsai, Keh-Sung; Yang, Rong-Sen; Liu, Shing-Hwa

    2014-04-01

    The risk of low birth weights is elevated in prenatal exposure to polycyclic aromatic hydrocarbons (PAHs), which are ubiquitous environmental pollutants generated from combustion of organic compounds, including cigarette smoke. We hypothesized that benzo(a)pyrene (BaP), a member of PAHs existing in cigarette smoke, may affect the myogenesis to cause low birth weights. We investigated the effects of BaP and its main metabolite, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), on the myogenic differentiation of human skeletal muscle-derived progenitor cells (HSMPCs). HSMPCs were isolated by a modified preplate technique and cultured in myogenic differentiation media with or without BaP and BPDE (0.25 and 0.5 μM) for 4 days. The multinucleated myotube formation was morphologically analyzed by hematoxylin and eosin staining. The expressions of myogenic differentiation markers and related signaling proteins were determined by Western blotting. Both BaP and BPDE at the submicromolar concentrations (0.25 and 0.5 μM) dose-dependently repressed HSMPCs myogenic differentiation without obvious cell toxicity. Both BaP and BPDE inhibited the muscle-specific protein expressions (myogenin and myosin heavy chain) and phosphorylation of Akt (a known modulator in myogenesis), which could be significantly reversed by the inhibitors for aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and nuclear factor (NF)-κB. BaP- and BPDE-activated NF-κB-p65 protein phosphorylation could also be attenuated by both AhR and ER inhibitors. The inhibitory effects of BaP and BPDE on myogenesis were reversed after withdrawing BaP exposure, but not after BPDE withdrawal. These results suggest that both BaP and BPDE are capable of inhibiting myogenesis via an AhR- or/and ER-regulated NF-κB/Akt signaling pathway.

  19. Puerarin offsets the anticoagulation effect of warfarin in rats by inducing rCyps, upregulating vitamin K epoxide reductase and inhibiting thrombomodulin.

    PubMed

    Ge, Beikang; Zhang, Zhen; Lam, Teddy Taining; Zuo, Zhong

    2017-01-01

    The current study was conducted to investigate the potential pharmacokinetic and pharmacodynamic interactions between warfarin and puerarin which is the most abundant component in Pueraria lobata (Gegen). In vivo and ex vivo rat models were used to reveal the underlying mechanisms of such interactions. Apart from one control group, five groups of Sprague-Dawley rats were treated with warfarin, oral puerarin, oral puerarin with warfarin, intravenous puerarin, intravenous puerarin with warfarin. The treatment lasted for 5 consecutive days. Thereafter, the levels of warfarin, warfarin metabolites and puerarin in plasma of these rats were monitored and compared. The rCyps activity and expression in rat livers of different treatment groups were assessed. The prothrombin time was observed. The vitamin K epoxide reductase (VKOR) activity and expression in rat livers were evaluated. Thrombomodulin activity and expression in the rat lung and rat plasma were assessed. The soluble thrombomodulin (sTM) concentrations of different treatment groups were examined. Intravenously administered puerarin altered the pharmacokinetics of warfarin significantly by shortening t1/2 , decreasing AUC0-96 h and increasing the clearance of warfarin. Further mechanistic studies suggested that both oral and intravenous administration of puerarin significantly induced the activities and expressions of rCyp2b1, rCyp2c6 and rCyp1a1. In addition, co-administration of puerarin reduced the prothrombin time of rat plasma by enhancing VKOR and inhibiting thrombomodulin. Puerarin increased warfarin metabolism and offset warfarin anticoagulation by inducing rCyps, upregulating VKOR and inhibiting thrombomodulin in rats. The clinical impact of such potential interactions warrants further verification. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  20. Inhibition of fatty acid amide hydrolase (FAAH) as a novel therapeutic strategy in the treatment of pain and inflammatory diseases in the gastrointestinal tract.

    PubMed

    Sałaga, Maciej; Sobczak, Marta; Fichna, Jakub

    2014-02-14

    Fatty acid amide hydrolase (FAAH) is the enzyme crucially involved in the modulation of physiological processes mediated by anandamide (AEA), as well as other endocannabinoids and non-cannabinoid biolipids in the gastrointestinal (GI) tract. FAAH also plays a major role in the etiology and the course of GI diseases and the inhibition of the enzyme has recently become a potential target for their therapy. In this review we look at the pharmacology of FAAH and possible clinical application of FAAH inhibitors in the treatment of GI disorders. In particular, we focus on inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS), whose symptoms include abdominal pain and motility disturbances. We also discuss why the inhibitor-based drugs may replace in future conventional therapies for IBD and IBS.

  1. High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker.

    PubMed

    Yapa, Udeni; Prusakiewicz, Jeffery J; Wrightstone, Ann D; Christine, Lori J; Palandra, Joe; Groeber, Elizabeth; Wittwer, Arthur J

    2012-02-15

    Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.

  2. Substrate specificity and inhibition of brassinin hydrolases, detoxifying enzymes from the plant pathogens Leptosphaeria maculans and Alternaria brassicicola.

    PubMed

    Pedras, M Soledade C; Minic, Zoran; Sarma-Mamillapalle, Vijay K

    2009-12-01

    Blackleg (Leptosphaeria maculans and Leptosphaeria biglobosa) and black spot (Alternaria brassicicola) fungi are devastating plant pathogens known to detoxify the plant defence metabolite, brassinin. The significant roles of brassinin as a crucifer phytoalexin and as a biosynthetic precursor of several other plant defences make it important in plant fitness. Brassinin detoxifying enzymes produced by L. maculans and A. brassicicola catalyse the detoxification of brassinin by hydrolysis of its dithiocarbamate group to indolyl-3-methanamine. The purification and characterization of brassinin hydrolases produced by L. maculans (BHLmL2) and A. brassicicola (BHAb) were accomplished: native BHLmL2 was found to be a tetrameric protein with a molecular mass of 220 kDa, whereas native BHAb was found to be a dimeric protein of 120 kDa. Protein characterization using LC-MS/MS and sequence alignment analyses suggested that both enzymes belong to the family of amidases with the catalytic Ser/Ser/Lys triad. Furthermore, chemical modification of BHLmL2 and BHAb with selective reagents suggested that the amino acid serine was involved in the catalytic activity of both enzymes. The overall results indicated that BHs have new substrate specificities with a new catalytic activity that can be designated as dithiocarbamate hydrolase. Investigation of the effect of various phytoalexins on the activities of BHLmL2 and BHAb indicated that cyclobrassinin was a competitive inhibitor of both enzymes. On the basis of pH dependence, sequence analyses, chemical modifications of amino acid residues and identification of headspace volatiles, a chemical mechanism for hydrolysis of the dithiocarbamate group of brassinin catalysed by BHLmL2 and BHAb is proposed. The current information should facilitate the design of specific synthetic inhibitors of these enzymes for plant treatments against blackleg and black spot fungal infections.

  3. Mechanism of inhibition of fatty acid amide hydrolase by sulfonamide-containing benzothiazoles: long residence time derived from increased kinetic barrier and not exclusively from thermodynamic potency.

    PubMed

    Tian, Gaochao; Paschetto, Kathy A; Gharahdaghi, Farzin; Gordon, Euan; Wilkins, Dee E; Luo, Xincai; Scott, Clay W

    2011-08-16

    Fatty acid amide hydrolase (FAAH) has emerged as a potential target for developing analgesic, anxiolytic, antidepressant, sleep-enhancing, and anti-inflammatory drugs, and tremendous efforts have been made to discover potent and selective inhibitors of FAAH. Most known potent FAAH inhibitors described to date employ covalent mechanisms, inhibiting the enzyme either reversibly or irreversibly. Recently, a benzothiazole-based analogue (1) has been described possessing a high potency against FAAH yet lacking a structural feature previously known to interact with FAAH covalently. However, covalent inhibition of FAAH by 1 has not been fully ruled out, and the issue of reversibility has not been addressed. Confirming previous reports, 1 inhibited recombinant human FAAH (rhFAAH) with high potency with IC(50) ~2 nM. It displayed an apparently noncompetitive and irreversible inhibition, titrating rhFAAH stoichiometrically within normal assay times. The inhibition appeared to be time dependent, but the time dependence only improved potency by a small degree (from ~8 to ~2 nM). However, mass spectrometric analyses of the reaction mixture failed to reveal any cleavage product or covalent adduct and showed full recovery of the parent compound, ruling out covalent, irreversible inhibition. Dialysis revealed recovery of enzyme activity from enzyme-inhibitor complex over a prolonged time (>10 h), demonstrating that 1 is indeed a reversible, albeit slowly dissociating inhibitor of FAAH. Molecular docking indicated that the sulfonamide group of 1 could form hydrogen bonds with several residues involved in catalysis, thereby mimicking the transition state. The long residence time displayed by 1 does not appear to derive exclusively from great thermodynamic potency and is consistent with an increased kinetic energy barrier that prevents dissociation from happening quickly.

  4. Teratogen metabolism: activation of thalidomide and thalidomide analogues to products that inhibit the attachment of cells to concanavalin A coated plastic surfaces. Revised version

    SciTech Connect

    Braun, A.G.; Weinreb, S.L.

    1982-01-01

    Thalidomide metabolites inhibit the attachment of tumor cells to concanavalin A coated polyethylene surfaces. Thalidomide, itself, is non-inhibitory. Thalidomide activation to inhibitory products requires hepatic microsomes, an NADPH generating system and molecular oxygen. Production of inhibitory metabolites is unaffected by either epoxide hydrolase or TCPO, an inhibitor of epoxide hydrolase endogenous to hepatic S9 fraction. Therefore the attachment inhibitor is probably not an arene oxide. Inhibition is not accompanied by cytotoxicity as judged by trypan blue exclusion. Although uninduced hepatic microsomes from mice, rats and dogs have similar ability to activate thalidomide, microsomes from Aroclor 1254 induced rats are relatively inactive in the system. Inhibitory metabolites can be generated from the thalidomide analogues EM8, EM12, EM16, EM87, EM136, EM255, E350, phthalimide, phthalimido-phthalimide, indan, 1-indanone and 1,3-indandione. Glutarimide, glutamic acid and phthalic acid do not activate to inhibitory products.

  5. Direct epoxidation in Candida antarctica lipase B studied by experiment and theory.

    PubMed

    Svedendahl, Maria; Carlqvist, Peter; Branneby, Cecilia; Allnér, Olof; Frise, Anton; Hult, Karl; Berglund, Per; Brinck, Tore

    2008-10-13

    Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated by density functional theory calculations, and it was demonstrated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

  6. Detoxication of the 2',3'-epoxide metabolites of allylbenzene and estragole. Conjugation with glutathione.

    PubMed

    Luo, G; Guenthner, T M

    1994-01-01

    The enzymatic detoxication in vitro of the 2',3'-epoxide derivatives of allylbenzene and estragole was examined, and the relative rates of enzymatic glutathione conjugation and epoxide hydrolysis were compared with those for styrene 1',2'-oxide. HPLC was used to determine the amounts of dihydrodiol and glutathione conjugate metabolites formed by cell extracts from several sources. Although some differences among species were observed, in general, the rates of epoxide inactivation by both pathways are similar. We conclude that one explanation for the apparent lack of genotoxicity of these allylic epoxides in vivo may be their rapid metabolic inactivation by both glutathione S-transferases and epoxide hydrolases, which occur to approximately equal degrees in vitro.

  7. Epoxidation catalyst and process

    DOEpatents

    Linic, Suljo; Christopher, Phillip

    2010-10-26

    Disclosed herein is a catalytic method of converting alkenes to epoxides. This method generally includes reacting alkenes with oxygen in the presence of a specific silver catalyst under conditions suitable to produce a yield of the epoxides. The specific silver catalyst is a silver nanocrystal having a plurality of surface planes, a substantial portion of which is defined by Miller indices of (100). The reaction is performed by charging a suitable reactor with this silver catalyst and then feeding the reactants to the reactor under conditions to carry out the reaction. The reaction may be performed in batch, or as a continuous process that employs a recycle of any unreacted alkenes. The specific silver catalyst has unexpectedly high selectivity for epoxide products. Consequently, this general method (and its various embodiments) will result in extraordinarily high epoxide yields heretofore unattainable.

  8. Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells.

    PubMed Central

    Di Marzo, V; Melck, D; Orlando, P; Bisogno, T; Zagoory, O; Bifulco, M; Vogel, Z; De Petrocellis, L

    2001-01-01

    Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB(1)). This synergistic effect was reduced by the CB(2) cannabinoid receptor antagonist SR144528, although PEA does not activate either CB(1) or CB(2) receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1-10 microM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 microM) decreased the IC(50) values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB(2) receptor antagonist SR144528, and was not mimicked by a selective agonist of CB(2) receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 microM PEA caused a approximately 30-40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB(1) or CB(2) receptors, and neither did it alter the CB(1)/CB(2)-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB(1) and CB(2) receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression. PMID:11485574

  9. Succinic anhydrides from epoxides

    DOEpatents

    Coates, Geoffrey W.; Rowley, John M.

    2013-07-09

    Catalysts and methods for the double carbonylation of epoxides are disclosed. Each epoxide molecule reacts with two molecules of carbon monoxide to produce a succinic anhydride. The reaction is facilitated by catalysts combining a Lewis acidic species with a transition metal carbonyl complex. The double carbonylation is achieved in single process by using reaction conditions under which both carbonylation reactions occur without the necessity of isolating or purifying the product of the first carbonylation.

  10. Succinic anhydrides from epoxides

    DOEpatents

    Coates, Geoffrey W.; Rowley, John M.

    2016-06-28

    Catalysts and methods for the double carbonylation of epoxides are disclosed. Each epoxide molecule reacts with two molecules of carbon monoxide to produce a succinic anhydride. The reaction is facilitated by catalysts combining a Lewis acidic species with a transition metal carbonyl complex. The double carbonylation is achieved in single process by using reaction conditions under which both carbonylation reactions occur without the necessity of isolating or purifying the product of the first carbonylation.

  11. Succinic anhydrides from epoxides

    DOEpatents

    Coates, Geoffrey W; Rowley, John M

    2014-12-30

    Catalysts and methods for the double carbonylation of epoxides are disclosed. Each epoxide molecule reacts with two molecules of carbon monoxide to produce a succinic anhydride. The reaction is facilitated by catalysts combining a Lewis acidic species with a transition metal carbonyl complex. The double carbonylation is achieved in single process by using reaction conditions under which both carbonylation reactions occur without the necessity of isolating or purifying the product of the first carbonylation.

  12. Inhibiting parabrachial fatty acid amide hydrolase activity selectively increases the intake of palatable food via cannabinoid CB1 receptors.

    PubMed

    Dipatrizio, Nicholas V; Simansky, Kenny J

    2008-11-01

    These studies investigated feeding responses to indirect activation of parabrachial cannabinoid CB1 receptors. Arachidonoyl serotonin (AA5HT), an inhibitor of the endocannabinoid degradative enzyme, fatty acid amide hydrolase (FAAH), was infused into the parabrachial nucleus of male Sprague-Dawley rats, and intakes of high-fat/sucrose pellets and standard rodent chow were subsequently evaluated under various feeding schedules. FAAH blockade stimulated the intake of high-fat/sucrose pellets that were presented daily for 4 h during the light period, with compensatory decreases in the consumption of standard chow during the ensuing 20 h. These diet-selective changes were repeated on the next day, indicating a shift in feeding toward the more palatable diet that lasted for 48 h after a single infusion. The cannabinoid CB1 receptor antagonist, AM251, blocked the orexigenic actions of AA5HT, implicating CB1 receptors in mediating the feeding responses to FAAH inactivation. When the feeding schedule was reversed, AA5HT produced nominal increases in the consumption of standard chow for the 4-h access period, but substantial increases in the intake of high-fat/sucrose during the following 20-h interval. When presented with only high-fat/sucrose diet for 24 h, AA5HT increased 24-h food intake. In contrast, when given 24-h access only to standard chow, AA5HT failed to affect intake. Therefore, indirectly activating parabrachial CB1 receptors by blocking the degradation of native ligands selectively stimulates the intake of palatable food, with differential actions on total energy intake depending upon the feeding schedule. Our results support a role for parabrachial cannabinoid mechanisms in providing physiological regulation to neural substrates modulating feeding, energy balance, and behavioral responses for natural reward.

  13. Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis.

    PubMed

    Bender-Machado, Lilia; Bäuerlein, Michael; Carrari, Fernando; Schauer, Nicolas; Lytovchenko, Anna; Gibon, Yves; Kelly, Amelie A; Loureiro, Marcello; Müller-Röber, Bernd; Willmitzer, Lothar; Fernie, Alisdair R

    2004-07-01

    Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

  14. Conformational diversity and enantioconvergence in potato epoxide hydrolase 1† †Electronic supplementary information (ESI) available: Further details on calibration of our simulations, QM data and RMSD plots from the MD simulations, further experimental data, and all EVB parameters used to model styrene oxide hydrolysis in this study. See DOI: 10.1039/c6ob00060f Click here for additional data file.

    PubMed Central

    Bauer, P.; Carlsson, Å. Janfalk; Amrein, B. A.

    2016-01-01

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio- and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio- and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis. PMID:27049844

  15. Inhibiting Inosine Hydrolase and Alanine Racemase to Enhance the Germination of Bacillus anthracis Sterne Spores: Potential Spore Decontamination Strategies

    DTIC Science & Technology

    2015-06-19

    Approved for public release; distribution is unlimited. UNCLASSIFIED PR-15-306 Anthrax Background ~’ . . . ’ ’ . . . ’ . . . . ’ . ’ • Caused by a gram ... positive spore forming rod. • Important veterinary disease as herbivores may be prone to the disease if they feed in ’anthrax zones...By inhibiting Air and lunH separately - By inhibiting the both concommitantly (iunH mutant spores positively affected by the block of Air) • Better

  16. Glycoside hydrolases having multiple hydrolase activities

    DOEpatents

    Chen, Zhiwei; Friedland, Gregory D.; Chhabra, Swapnil R.; Chivian, Dylan C.; Simmons, Blake A

    2017-08-08

    Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

  17. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

    PubMed

    Vandevoorde, Séverine; Fowler, Christopher J

    2005-08-01

    There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for

  18. The Role of Long Chain Fatty Acids and Their Epoxide Metabolites in Nociceptive Signaling

    PubMed Central

    Wagner, Karen; Vito, Steve; Inceoglu, Bora; Hammock, Bruce D.

    2014-01-01

    Lipid derived mediators contribute to inflammation and the sensing of pain. The contributions of omega-6 derived prostanoids in enhancing inflammation and pain sensation are well known. Less well explored are the opposing anti-inflammatory and analgesic effects of the omega-6 derived epoxyeicosatrienoic acids. Far less has been described about the epoxidized metabolites derived from omega-3 long chain fatty acids. The epoxide metabolites are turned over rapidly with enzymatic hydrolysis by the soluble epoxide hydrolase being the major elimination pathway. Despite this, the overall understanding of the role of lipid mediators in the pathology of chronic pain is growing. Here we review the role of long chain fatty acids and their metabolites in alleviating both acute and chronic pain conditions. We focus specifically on the epoxidized metabolites of omega-6 and omega-3 long chain fatty acids as well as a novel strategy to modulate their activity in vivo. PMID:25240260

  19. A multi-target approach for pain treatment: dual inhibition of fatty acid amide hydrolase and TRPV1 in a rat model of osteoarthritis.

    PubMed

    Malek, Natalia; Mrugala, Monika; Makuch, Wioletta; Kolosowska, Natalia; Przewlocka, Barbara; Binkowski, Marcin; Czaja, Martyna; Morera, Enrico; Di Marzo, Vincenzo; Starowicz, Katarzyna

    2015-05-01

    The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

  20. The putative α/β-hydrolases of Dietzia cinnamea P4 strain as potential enzymes for biocatalytic applications.

    PubMed

    Procópio, Luciano; Macrae, Andrew; van Elsas, Jan Dirk; Seldin, Lucy

    2013-03-01

    The draft genome of the soil actinomycete Dietzia cinnamea P4 reveals a versatile group of α/β-hydrolase fold enzymes. Phylogenetic and comparative sequence analyses were used to classify the α/β-hydrolases of strain P4 into six different groups: (i) lipases, (ii) esterases, (iii) epoxide hydrolases, (iv) haloacid dehalogenases, (v) C-C breaking enzymes and (vi) serine peptidases. The high number of lipases/esterases (41) and epoxide hydrolase enzymes (14) present in the relatively small (3.6 Mb) P4 genome is unusual; it is likely to be linked to the survival of strain P4 in its natural environment. Strain P4 is thus equipped with a large number of genes which would appear to confer survivability in harsh hot tropical soil. As such, this highly resilient soil bacterial strain provides an interesting genome for enzyme mining for applications in the field of biotransformations of polymeric compounds.

  1. Microbial production of epoxides

    DOEpatents

    Clark, Thomas R.; Roberto, Francisco F.

    2003-06-10

    A method for microbial production of epoxides and other oxygenated products is disclosed. The method uses a biocatalyst of methanotrophic bacteria cultured in a biphasic medium containing a major amount of a non-aqueous polar solvent. Regeneration of reducing equivalents is carried out by using endogenous hydrogenase activity together with supplied hydrogen gas. This method is especially effective with gaseous substrates and cofactors that result in liquid products.

  2. Epoxide-derived organosulfates

    NASA Astrophysics Data System (ADS)

    Schwier, A. N.; Woo, J.; McNeill, V. F.

    2011-12-01

    Organosulfates (OS) are a significant fraction of secondary organic aerosol (SOA) material in the atmosphere. OS are typically surface-active, and have been suggested to cause surface tension depression in aerosols. Recent field studies suggest that epoxide-derived OS are the most abundant OS type in aerosols. Time-dependent surface tension measurements and Aerosol-CIMS characterization of two epoxides and their organosulfate products are shown. α-pinene oxide, derived from α-pinene, shows significant surface tension depression in H2O and ammonium sulfate. Results from cis-2,3-epoxybutane-1,4-diol (BEPOX), a butadiene-derived analog to isoprene-derived epoxydiols, are also shown. In addition, using GAMMA, a photochemical box model using coupled gas- and aqueous-phase chemistry developed in the McNeill laboratory, we show the dominance of epoxide-derived OS formation over other competing OS formation mechanisms, such as radical chemistry, under both high-NOx and low-NOx scenarios.

  3. Expression of fatty acid amide hydrolase (FAAH) in human, mouse, and rat urinary bladder and effects of FAAH inhibition on bladder function in awake rats.

    PubMed

    Strittmatter, Frank; Gandaglia, Giorgio; Benigni, Fabio; Bettiga, Arianna; Rigatti, Patrizio; Montorsi, Francesco; Gratzke, Christian; Stief, Christian; Colciago, Giorgia; Hedlund, Petter

    2012-01-01

    Cannabinoid receptor (CB)-mediated functions may be involved in the regulation of bladder function, but information on endocannabinoid signals during micturition is scarce. Investigate the expression of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in human, rat, and mouse bladders and study the effects of inhibition of FAAH during urodynamics in awake rats. Bladder tissue from humans, mice, and rats was used for measurements. Female Sprague-Dawley rats were administered the FAAH inhibitor oleoyl ethyl amide (OEtA) or vehicle intravenously (IV) or intravesically (IVES) with or without rimonabant (CB1 antagonist) or SR144528 (CB2 antagonist). Real-time transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and cystometry in awake rats. Messenger RNA and protein for FAAH was expressed in the mucosa of human, mouse, and rat urinary bladders. Immunoreactivities for FAAH and CB2 were codistributed in rat and human urothelium. IV OEtA (0.3mg/kg) to rats increased intercontraction intervals (ICIs), micturition volume (MV), bladder capacity (BC), and threshold pressure (TP) by 17±1%, 16±1%, 17±1%, and 19±5%, respectively (all p<0.05 vs baseline). IVES OEtA (1 and 10mg/l) in rats dose-dependently increased (p<0.05 vs baseline) ICI (19±2% and 35±5%), MV (15±3% and 32±4%), BC (16±2% and 34±4%), and TP (15±1%, 21±3%). SR144528 (IVES 5mg/l) abolished all effects of OEtA, whereas rimonabant only counteracted effects of OEtA on TP. Bladder mucosa of all species expressed FAAH. Rat and human urothelium coexpressed FAAH and CB2. The FAAH inhibitor OEtA altered urodynamic parameters that reflect sensory functions of micturition in rats. Suggesting a role for the endocannabinoid system in bladder mechanoafferent functions of rats, effects of IVES OEtA were abolished by an IVES CB2 antagonist and partly counteracted by an IVES CB1 antagonist. Copyright © 2011 European Association of Urology. Published by Elsevier B.V. All

  4. Friction reducing properties and stability of epoxidized oleochemicals

    USDA-ARS?s Scientific Manuscript database

    We have studied the properties of epoxidized oleochemical methyl esters including epoxidized soybean oil, epoxidized methyl oleate, epoxidized methyl linoleate, and epoxidized methyl linolenate. We have compared these materials to a similar series of unmodified olefins. Several interesting trends ...

  5. Mutagenicity of chloroalkene epoxides in bacterial systems.

    PubMed

    Kline, S A; McCoy, E C; Rosenkranz, H S; Van Duuren, B L

    1982-04-01

    6 alpha-chloroepoxides have been tested for in vitro activity in a variety of systems. The epoxides were cis- and trans-1-chloropropene oxide, cis- and trans-1,3-dichloropropene oxide, trichloroethylene oxide and tetrachloroethylene oxide. The epoxides were assayed for mutagenicity in the absence of metabolic activation in S. typhimurium TA1535 and E. coli WP2 uvrA and for preferential inhibition of growth of DNA-repair-deficient E. coli. All 6 epoxides possessed DNA-modifying activity as evidenced by their ability to preferentially inhibit DNA polymerase-deficient E. coli. All of the test chemicals except trichloroethylene oxide were mutagenic for S. typhimurium and all except trichloroethylene oxide and tetrachloroethylene oxide were mutagenic for E. coli Wp2 uvrA. Cis- and trans-1,3-dichloropropene oxide were the most potent mutagens and DNA modifiers. For all cases, the cis isomers were more active than the corresponding trans isomers. alpha-Chloroepoxides are considered likely to be the active intermediates of the carcinogenic parent halo-olefins. These mutagenicity studies are considered relevant in assessing the carcinogenicity of the parent hydrocarbons.

  6. Identification of cyanidin glycosides as constituents of freeze-dried black raspberries which inhibit anti-benzo[a]pyrene-7,8-diol-9,10-epoxide induced NFκB and AP-1 activity

    PubMed Central

    Hecht, Stephen S.; Huang, Chuanshu; Stoner, Gary D.; Li, Jingxia; Kenney, Patrick M. J.; Sturla, Shana J.; Carmella, Steven G.

    2010-01-01

    Dietary freeze-dried black raspberries inhibit tumor induction by N-nitrosomethylbenzylamine in the rat esophagus, but the constituents responsible for this chemopreventive activity have not been identified. We fractionated freeze-dried black raspberries and used mouse epidermal JB6 Cl 41 cells stably transfected with either a nuclear factor kappa B (NFκB)- or an activator protein 1 (AP-1)-luciferase reporter, and treated with racemic anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), to assess the inhibitory effects of the fractions. The ethanol and water extracts of the freeze-dried black raspberries had inhibitory activity and these extracts were fractionated by HPLC to give several bioactive fractions. Further HPLC analysis yielded multiple subfractions, some of which inhibited BPDE-induced NFκB activity. Major constituents of the most active subfractions were identified by their spectral properties and in comparison with standards as cyanidin-3-O-glucoside, cyanidin 3-O-(2G-xylosylrutinoside) and cyanidin 3-O-rutinoside. Analysis of freeze-dried black raspberries indicated that these three components comprised ∼3.4% of the material by dry weight. Consistent with these results, standard cyanidin-3-O-glucoside and cyanidin chloride were also good inhibitors of BPDE-induced NFκB activity. The results of this study demonstrate that cyanidin glycosides of freeze-dried black raspberries are bioactive compounds which could account for at least some of the chemopreventive activity observed in animal models. PMID:16522666

  7. Benzo[a]pyrene-7,8-diol-9,10-epoxide inhibits gap junction intercellular communication via phosphorylation of tumor progression locus 2 in WB-F344 rat liver epithelial cells.

    PubMed

    Lee, Bo Kyung; Chung, Min-Yu; Lee, Ki Won

    2015-05-01

    Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), a major metabolite of benzo[a]pyrene, has been reported to function as a human carcinogen. However, the molecular mechanism of how B[a]PDE regulates signaling pathways during tumor promotion remains unclear. In this study, we investigated the effects of B[a]PDE on the regulation of gap junction intercellular communication (GJIC), one of the major carcinogenic processes, and its main regulatory signaling pathways using WB-F344 rat liver epithelial (WB-F344 RLE) cells. Treatment of benzo[a]pyrene or B[a]PDE resulted in GJIC inhibition, and B[a]PDE was more active at lower concentrations than benzo[a]pyrene in the suppression of GJIC. This suggests that B[a]PDE is a stronger GJIC inhibitor. B[a]PDE at 1 µM reversibly inhibited GJIC in WB-F344 RLE cells, which was attributable to hyperphosphorylation of connexin43 (Cx43) via phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). We found that B[a]PDE induced phosphorylation of tumor progression locus 2 (Tpl2), a direct upstream regulator of MEK. Tpl2 inhibitor recovered B[a]PDE-induced GJIC inhibition and attenuated B[a]PDE-induced MEK/ERK phosphorylation in WB-F344 RLE cells. Collectively, our results suggest that B[a]PDE suppresses GJIC by activating Tpl2 and subsequently the MEK/ERK pathway and Cx43 phosphorylation in WB-F344 RLE cells. These results outline the potential importance of Tpl2 as a novel therapeutic target for B[a]PDE-induced GJIC inhibition during cancer promotion.

  8. A mechanistic study into the epoxidation of carboxylic acid and alkene in a mono, di-acylglycerol lipase.

    PubMed

    Wang, Xuping; Tang, Qingyun; Popowicz, Grzegorz Maria; Yang, Bo; Wang, Yonghua

    2015-05-01

    More and more industrial chemistry reactions rely on green technologies. Enzymes are finding increasing use in diverse chemical processes. Epoxidized vegetable oils have recently found applications as plasticizers and additives for PVC production. We report here an unusual activity of the Malassezia globosa lipase (SMG1) that is able to catalyze epoxidation of alkenes. SMG1 catalyzes formation of peroxides from long chain carboxylic acids that subsequently react with double bonds of alkenes to produce epoxides. The SMG1 is selective towards carboxylic acids and active also as a mutant lacking hydrolase activity. Moreover we present previously unobserved mechanism of catalysis that does not rely on acyl-substrate complex nor tetrahedral intermediate. Since SMG1 lipase is activated by allosteric change upon binding to the lipophilic-hydrophilic phase interface we reason that it can be used to drive the epoxidation in the lipophilic phase exclusively.

  9. Inverting hydrolases and their use in enantioconvergent biotransformations

    PubMed Central

    Schober, Markus; Faber, Kurt

    2013-01-01

    Owing to the more abundant occurrence of racemic compounds compared to prochiral or meso forms, most enantiomerically pure products are obtained via racemate resolution. This review summarizes (chemo)enzymatic enantioconvergent processes based on the use of hydrolytic enzymes, which are able to invert a stereocenter during catalysis that can overcome the 50%-yield limitation of kinetic resolution. Recent developments are presented in the fields of inverting or retaining sulfatases, epoxide hydrolases and dehalogenases, which allow the production of secondary alcohols or vicinal diols at a 100% theoretical yield from a racemate via enantioconvergent processes. PMID:23809848

  10. The endogenous cannabinoid anandamide produces delta-9-tetrahydrocannabinol-like discriminative and neurochemical effects that are enhanced by inhibition of fatty acid amide hydrolase but not by inhibition of anandamide transport.

    PubMed

    Solinas, Marcello; Tanda, Gianluigi; Justinova, Zuzana; Wertheim, Carrie E; Yasar, Sevil; Piomelli, Daniele; Vadivel, Subramanian K; Makriyannis, Alexandros; Goldberg, Steven R

    2007-04-01

    Anandamide is an endogenous ligand for brain cannabinoid CB(1) receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that anandamide, given i.v. or i.p., would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methanandamide (AM-356), a metabolically stable anandamide analog, produced clear dose-related THC-like discriminative effects, but anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of anandamide, with 3 mg/kg anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of anandamide after URB-597 and methanandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of anandamide or its dopamine-elevating effects. Thus, anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting

  11. Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth.

    PubMed

    Travers, Marie-Agnès; Sow, Cissé; Zirah, Séverine; Deregnaucourt, Christiane; Chaouch, Soraya; Queiroz, Rayner M L; Charneau, Sébastien; Allain, Thibault; Florent, Isabelle; Grellier, Philippe

    2016-01-01

    Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

  12. Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

    PubMed Central

    Travers, Marie-Agnès; Sow, Cissé; Zirah, Séverine; Deregnaucourt, Christiane; Chaouch, Soraya; Queiroz, Rayner M. L.; Charneau, Sébastien; Allain, Thibault; Florent, Isabelle; Grellier, Philippe

    2016-01-01

    Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present. PMID:27729900

  13. Activity of murein hydrolases in synchronized cultures of Escherichia coli.

    PubMed Central

    Hakenbeck, R; Messer, W

    1977-01-01

    Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited. PMID:321419

  14. Epoxide-mediated differential packaging of Cif and other virulence factors into outer membrane vesicles.

    PubMed

    Ballok, Alicia E; Filkins, Laura M; Bomberger, Jennifer M; Stanton, Bruce A; O'Toole, George A

    2014-10-01

    Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. Epoxide-Mediated Differential Packaging of Cif and Other Virulence Factors into Outer Membrane Vesicles

    PubMed Central

    Ballok, Alicia E.; Filkins, Laura M.; Bomberger, Jennifer M.; Stanton, Bruce A.

    2014-01-01

    Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. PMID:25112474

  16. Photopolymerizations of multicomponent epoxide and acrylate/epoxide hybrid systems for controlled kinetics and enhanced material properties

    NASA Astrophysics Data System (ADS)

    Eom, Ho Seop

    2011-12-01

    . These conclusions are consistent with physical property results. The enhanced fracture toughness and impact resistance were attributed to multimodal network chain-length distribution of copolymers containing the oligomer content between 70% and 80%. For acrylate/epoxide hybrid mixtures, diacrylate oligomers significantly suppressed reactivities of cycloaliphatic mono/diepoxides, which was attributed to high mixture viscosity and highly crosslinked acrylate network. In this case, the dual photoinitiator system did not favor the epoxide reaction. Depending on the monovinyl acrylate secondary functionalities, enhanced reactivity and ultimate conversion of the diepoxide were attributed to a combined effect of a reduced viscosity and the radical-promoted cationic polymerization associated with the dual photoinitiator. The retarded and inhibited diepoxide reactivities with ether and urethane secondary groups were attributed to solvation and nucleophilicity/basicity effects, respectively. The influence of the diepoxide on the acrylate reactivity was attributed to dilution and polarity effects. In this case, high concentration of the free-radical photoinitiator is required for the dual photoinitiator system. Physical properties of hybrid polymers also varied with acrylate structures and monomer composition. Dynamic modulation methods were proposed to enhance the diepoxide reactivity and final properties in the presence of urethane acrylates.

  17. [Protective effect of tiacalix[4]arene-tetrasulphonate on heavy metal inhibition of myometrium myosin subfragment-1 ATP-hydrolase activity].

    PubMed

    Labyntseva, R D; Bevza, O V; Bevza, A A; Liul'ko, A O; Kharchenko, S H; Kal'chenko, V I; Kosterin, S O

    2014-01-01

    Heavy metals have a negative effect on the contractility of uterine smooth muscles (myometrium), these effects can lead to various pathologies of a women reproductive system. To overcome these effects the methods for correcting the myometrium contractile activity are to be developed. Catalyzed by myosin ATPase ATP hydrolysis is the most important reaction in the molecular mechanism of myometrium contraction. We have found an inhibitory effect of 0.03-0.3 mM Ni2+, Pb2+ and Cd2+ on enzymatic hydrolysis of ATP by myosin subfragment-1 obtained from swine uterine smooth muscles. We have demonstrated that 100 μM thiacalix[4]arene-tetrasulphonate (C-798) recovered to the control level of ATPase activity of myosin subfragment-1 in the presence of heavy metal cations. One of the most probable mechanisms of C-798 corrective activity is based on its ability to chelate heavy metals, thus cations Pb, Cd and Ni can be removed from the incubation medium. Computer simulation has demonstrated that the protective effect of C-798 may also be the result of weakening the interaction of heavy metal ions with amino acid residues of the myosin molecule near the active site of ATP hydrolase. The obtained results can be used for further research aimed at assessing the prospects of thiacalix[4]arene-tetrasulfonate as pharmacological compounds.

  18. Inhibition of fatty-acid amide hydrolase enhances cannabinoid stress-induced analgesia: sites of action in the dorsolateral periaqueductal gray and rostral ventromedial medulla.

    PubMed

    Suplita, Richard L; Farthing, Jesse N; Gutierrez, Tannia; Hohmann, Andrea G

    2005-12-01

    Recent research in our laboratory has demonstrated that stress activates an endogenous cannabinoid mechanism that suppresses sensitivity to pain [Nature 435 (2005) 1108]. In this work, CB(1) antagonists administered systemically blocked stress-induced analgesia induced by brief, continuous foot-shock. The present studies were conducted to examine the role of cannabinoid CB(1) receptors in the brainstem rostral ventromedial medulla (RVM) and midbrain dorsolateral periaqueductal gray (PAG) in cannabinoid stress-induced analgesia (SIA). Pharmacological blockade of vanilloid TRPV1 receptors with capsazepine, administered systemically, did not alter cannabinoid SIA, suggesting that cannabinoid SIA was not dependent upon TRPV1. Microinjection of the competitive CB(1) antagonist rimonabant (SR141716A) into either the RVM or dorsolateral PAG suppressed stress antinociception in this model. Rimonabant was maximally effective following microinjection into the dorsolateral PAG. The fatty-acid amide hydrolase (FAAH) inhibitor arachidonoyl serotonin (AA-5-HT) was subsequently used to block hydrolysis of endocannabinoids and enhance SIA. Systemic and site-specific injections of AA-5-HT into either the dorsolateral PAG or RVM induced CB(1)-mediated enhancements of SIA. Palmitoyltrifluoromethylketone, a potent inhibitor of FAAH and phospholipase A2 activity, administered systemically, exerted similar effects. In all conditions, the antinociceptive effects of each FAAH inhibitor were completely blocked by coadministration of the CB(1) antagonist rimonabant. The present results provide evidence that a descending cannabinergic neural system is activated by environmental stressors to modulate pain sensitivity in a CB(1)-dependent manner.

  19. S-Mercuration of ubiquitin carboxyl-terminal hydrolase L1 through Cys152 by methylmercury causes inhibition of its catalytic activity and reduction of monoubiquitin levels in SH-SY5Y cells.

    PubMed

    Toyama, Takashi; Abiko, Yumi; Katayama, Yuko; Kaji, Toshiyuki; Kumagai, Yoshito

    2015-12-01

    Methylmercury (MeHg) is an environmental electrophile that covalently modifies cellular proteins. In this study, we identified proteins that undergo S-mercuration by MeHg. By combining two-dimensional SDS-PAGE, atomic absorption spectrometry and ultra performance liquid chromatography mass spectrometry (UPLC/MS/MS), we revealed that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a target for S-mercuration in human neuroblastoma SH-SY5Y cells exposed to MeHg (1 µM, 9 hr). The modification site of UCH-L1 by MeHg was Cys152, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. MeHg was shown to inhibit the catalytic activity of recombinant human UCH-L1 in a concentration-dependent manner. Knockdown of UCH-L1 indicated that this enzyme plays a critical role in regulating mono-ubiquitin (monoUb) levels in SH-SY5Y cells and exposure of SH-SY5Y cells to MeHg caused a reduction in the level of monoUb in these cells. These observations suggest that UCH-L1 readily undergoes S-mercuration by MeHg through Cys152 and this covalent modification inhibits UCH-L1, leading to the potential disruption of the maintenance of cellular monoUb levels.

  20. Molecular cloning and amino acid sequence of leukotriene A4 hydrolase.

    PubMed Central

    Funk, C D; Rådmark, O; Fu, J Y; Matsumoto, T; Jörnvall, H; Shimizu, T; Samuelsson, B

    1987-01-01

    A cDNA clone corresponding to leukotriene A4 hydrolase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antiserum. Several additional clones from human lung and placenta cDNA lambda g11 libraries were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. One of these clones has an insert of 1910 base pairs that contains the complete protein-coding region. From the deduced primary structure, leukotriene A4 hydrolase is a 610 amino and protein with a calculated molecular weight of 69,140. No apparent homologies with microsomal epoxide hydrolases were found. RNA blot analysis indicated substantial amounts of a discrete mRNA of approximately equal to 2250 nucleotides in lung tissue and leukocytes. Images PMID:2821541

  1. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2017-07-11

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  2. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2013-02-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  3. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  4. 1-Heteroaryl-3-phenoxypropan-2-ones as inhibitors of cytosolic phospholipase A₂α and fatty acid amide hydrolase: Effect of the replacement of the ether oxygen with sulfur and nitrogen moieties on enzyme inhibition and metabolic stability.

    PubMed

    Sundermann, Tom; Fabian, Jörg; Hanekamp, Walburga; Lehr, Matthias

    2015-05-15

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that certain 3-phenoxy-substituted 1-heteroarylpropan-2-ones are inhibitors of cPLA2α and/or FAAH. Starting from 1-[2-oxo-3-(4-phenoxyphenoxy)propyl]indole-5-carboxylic acid (3) and 1-(1H-benzotriazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-one (4), the effect of the replacement of the oxygen in position 3 of the propan-2-one scaffold by sulfur and nitrogen containing moieties on inhibition of cPLA2α and fatty acid amide hydrolase as well as on metabolic stability in rat liver S9 fractions was investigated. As a result of these structure-activity relationship studies it was found that the ether oxygen is of great importance for enzyme inhibitory potency. Replacement by sulfur led to an about 100-fold decrease of enzyme inhibition, nitrogen and substituted nitrogen atoms at this position even resulted in inactivity of the compounds. The effect of the structural variations performed on metabolic stability of the important ketone pharmacophore was partly different in the two series of compounds. While introduction of SO and SO2 significantly increased stability of the ketone against reduction in case of the indole-5-carboxylic acid 3, it had no effect in case of the benzotriazole 4. Further analysis of the metabolism of 3 and 4 in rat liver S9 fractions revealed that the major metabolite of 3 was the alcohol 53 formed by reduction of the keto group. In contrast, in case of 4 beside keto reduction an excessive hydroxylation of the terminal phenoxy group occurred leading to the dihydroxy compound 50. Experiments with enzyme inhibitors showed that the phenylhydroxylation of 4 was catalyzed by tranylcypromine sensitive cytochrome P450 isoforms, while the reduction of the ketone function of 3 and 4 was mainly caused by cytosolic short chain dehydrogenases

  5. Effects of fatty acid amide hydrolase inhibition on neuronal responses to nicotine, cocaine and morphine in the nucleus accumbens shell and ventral tegmental area: involvement of PPAR-alpha nuclear receptors.

    PubMed

    Luchicchi, Antonio; Lecca, Salvatore; Carta, Stefano; Pillolla, Giuliano; Muntoni, Anna L; Yasar, Sevil; Goldberg, Steven R; Pistis, Marco

    2010-07-01

    The endocannabinoid system regulates neurotransmission in brain regions relevant to neurobiological and behavioral actions of addicting drugs. We recently demonstrated that inhibition by URB597 of fatty acid amide hydrolase (FAAH), the main enzyme that degrades the endogenous cannabinoid N-acylethanolamine (NAE) anandamide and the endogenous non-cannabinoid NAEs oleoylethanolamide and palmitoylethanolamide, blocks nicotine-induced excitation of ventral tegmental area (VTA) dopamine (DA) neurons and DA release in the shell of the nucleus accumbens (ShNAc), as well as nicotine-induced drug self-administration, conditioned place preference and relapse in rats. Here, we studied whether effects of FAAH inhibition on nicotine-induced changes in activity of VTA DA neurons were specific for nicotine or extended to two drugs of abuse acting through different mechanisms, cocaine and morphine. We also evaluated whether FAAH inhibition affects nicotine-, cocaine- or morphine-induced actions in the ShNAc. Experiments involved single-unit electrophysiological recordings from DA neurons in the VTA and medium spiny neurons in the ShNAc in anesthetized rats. We found that URB597 blocked effects of nicotine and cocaine in the ShNAc through activation of both surface cannabinoid CB1-receptors and alpha-type peroxisome proliferator-activated nuclear receptor. URB597 did not alter the effects of either cocaine or morphine on VTA DA neurons. These results show that the blockade of nicotine-induced excitation of VTA DA neurons, which we previously described, is selective for nicotine and indicate novel mechanisms recruited to regulate the effects of addicting drugs within the ShNAc of the brain reward system.

  6. Surprising unreactivity of cholesterol-5,6-epoxides towards nucleophiles[S

    PubMed Central

    Paillasse, Michael R.; Saffon, Nathalie; Gornitzka, Heinz; Silvente-Poirot, Sandrine; Poirot, Marc; de Medina, Philippe

    2012-01-01

    We recently established that drugs used for the treatment and the prophylaxis of breast cancers, such as tamoxifen, were potent inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), which led to the accumulation of 5,6α-epoxy-cholesterol (5,6α-EC) and 5,6β-epoxy-cholesterol (5,6β-EC). This could be considered a paradox because epoxides are known as alkylating agents with putative carcinogenic properties. We report here that, as opposed to the carcinogen styrene-oxide, neither of the ECs reacted spontaneously with nucleophiles. Under catalytic conditions, 5,6β-EC remains unreactive whereas 5,6α-EC gives cholestan-3β,5α-diol-6β-substituted compounds. These data showed that 5,6-ECs are stable epoxides and unreactive toward nucleophiles in the absence of a catalyst, which contrasts with the well-known reactivity of aromatic and aliphatic epoxides. These data rule out 5,6-EC acting as spontaneous alkylating agents. In addition, these data support the existence of a stereoselective metabolism of 5,6α-EC. PMID:22285872

  7. Epoxidation of Short-Chain Alkenes by Resting-Cell Suspensions of Propane-Grown Bacteria

    PubMed Central

    Hou, Ching T.; Patel, Ramesh; Laskin, Allen I.; Barnabe, Nancy; Barist, Irene

    1983-01-01

    Sixteen new cultures of propane-utilizing bacteria were isolated from lake water from Warinanco Park, Linden, N.J. and from lake and soil samples from Bayway Refinery, Linden, N.J. In addition, 19 known cultures obtained from culture collections were also found to be able to grow on propane as the sole carbon and energy source. In addition to their ability to oxidize n-alkanes, resting-cell suspensions of both new cultures and known cultures grown on propane oxidize short-chain alkenes to their corresponding 1,2-epoxides. Among the substrate alkenes, propylene was oxidized at the highest rate. In contrast to the case with methylotrophic bacteria, the product epoxides are further metabolized. Propane and other gaseous n-alkanes inhibit the epoxidation of propylene. The optimum conditions for in vivo epoxidation are described. Results from inhibition studies indicate that a propane monooxygenase system catalyzes both the epoxidation and hydroxylation reactions. Experiments with cell-free extracts show that both hydroxylation and epoxidation activities are located in the soluble fraction obtained after 80,000 × g centrifugation. PMID:16346338

  8. Alpha/Beta-hydrolase fold enzymes: structures, functions and mechanisms.

    PubMed

    Holmquist, M

    2000-09-01

    The alpha/beta-hydrolase fold family of enzymes is rapidly becoming one of the largest group of structurally related enzymes with diverse catalytic functions. Members in this family include acetylcholinesterase, dienelactone hydrolase, lipase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, hydroxynitrile lyase and others. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. For example, acetylcholine esterase catalyzes the cleavage of the neurotransmitter acetylcholine, at a rate close to the limits of diffusion of substrate to the active site of the enzyme. Dienelactone hydrolase uses substrate-assisted catalysis to degrade aromatic compounds. Lipases act adsorbed at the water/lipid interface of their neutral water-insoluble ester substrates. Most lipases have their active site buried under secondary structure elements, a flap, which must change conformation to allow substrate to access the active site. Thioesterases are involved in a multitude of biochemical processes including bioluminiscence, fatty acid- and polyketide biosynthesis and metabolism. Serine carboxypeptidases recognize the negatively charged carboxylate terminus of their peptide substrates. Haloalkane dehalogenase is a detoxifying enzyme that converts halogenated aliphatics to the corresponding alcohols, while haloperoxidase catalyzes the halogenation of organic compounds. Hydroxynitrile lyase cleaves carbon-carbon bonds in cyanohydrins with concomitant hydrogen cyanide formation as a defense mechanism in plants. This paper gives an overview of catalytic activities reported for this family of enzymes by discussing selected examples. The current state of knowledge of the molecular basis for catalysis and substrate specificity is outlined

  9. An unprecedented reversible mode of action of β-lactams for the inhibition of human fatty acid amide hydrolase (hFAAH).

    PubMed

    Feledziak, Marion; Michaux, Catherine; Lambert, Didier M; Marchand-Brynaert, Jacqueline

    2013-02-01

    A series of compound was prepared to clarify the reversible mechanism of β-lactamic hFAAH inhibitors on the one hand, and to modulate some of their physicochemical parameters on the other hand. In particular, two compounds (4b and 4e) were designed to display a potential good leaving group on the crucial carbonyl with a view to possibly acylating the active serine of the hFAAH catalytic triad. Reversibility studies showed that these two compounds retain the reversible mode of inhibition, suggesting a noncovalent interaction between our β-lactams and hFAAH. Finally, pharmacological evaluations of bioisosteres of the lead compound (4a, IC(50) = 5.3 nM) revealed that log P values and PSA could be optimized without altering the FAAH inhibition (IC(50) values from 3.65 nM to 70.9 nM). Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  10. Role of a novel soluble nucleotide phospho-hydrolase from sheep plasma in inhibition of platelet reactivity: hemostasis, thrombosis, and vascular biology.

    PubMed

    Birk, Alex V; Bubman, Darya; Broekman, M Johan; Robertson, Hugh D; Drosopoulos, Joan H F; Marcus, Aaron J; Szeto, Hazel H

    2002-02-01

    Ecto- and exoenzymes that metabolize extracellular adenosine diphosphate (ADP), the major promoter of platelet activation and recruitment, are of potential clinical importance because they can metabolically prevent excessive thrombus growth. An ecto-ADPase (CD39, NTPDase1) has been identified on endothelial cells. We demonstrate that ADP and adenosine triphosphate (ATP) are rapidly metabolized to adenosine monophosphate (AMP) in sheep plasma at pH 7.4. This hydrolysis is sensitive to P(1), P(5)-di-(adenosine-5') pentaphosphate (Ap(5)A), and ethylene glycol bis (beta-aminoethyl ether) - N, N, N(-), N(-) tetra-acetate (EGTA) but insensitive to tetramisole (an alkaline phosphatase inhibitor). A specific phosphodiesterase substrate, p -nitrophenol-5'-thymidine monophosphate (TMP) (p -Nph-5'-TMP), was readily hydrolyzed in sheep plasma at a rate of approximately 0.25 nmol/min/mg protein, and this hydrolysis was inhibited by ADP, ATP, and Ap(5)A. Furthermore, 200-fold purified p -Nph-5'-TMP-hydrolyzing activity also hydrolyzed ATP and ADP directly to AMP. When ADP was preincubated in plasma, its ability to induce platelet aggregation was inhibited in a time-dependent manner. This effect was abolished by Ap(5)A. The inhibitory effects on platelet aggregation correlated with hydrolysis of the ADP in plasma. These data suggest that the endogenous soluble plasma phosphohydrolase metabolizes ATP and ADP by means of cleavage of the alpha-beta-phosphodiester bond of nucleoside 5'-phosphate derivatives. This novel biochemical activity inhibits platelet reactivity through hydrolysis of extracellular nucleotides released by activated platelets during (patho)physiological processes, serving a homeostatic and antithrombotic function in vivo.

  11. Peptidoglycan Hydrolases of Escherichia coli

    PubMed Central

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  12. Acute and chronic toxicity toward the bacteria Vibrio fischeri of organic narcotics and epoxides: structural alerts for epoxide excess toxicity.

    PubMed

    Blaschke, Ulrike; Paschke, Albrecht; Rensch, Ines; Schüürmann, Gerrit

    2010-12-20

    The acute and chronic bacterial toxicity of 34 organic compounds comprising 19 baseline narcotics and 15 epoxides has been determined with regard to 30-min bioluminescence and 24-h growth inhibition in terms of EC50 (effective concentration 50%) values employing Vibrio fischeri. For the narcotics, linear regression of log EC50 on log Kow (octanol/water partition coefficient) yields r2 (squared correlation coefficient) and rms (root-mean-square error) values of 0.95 and 0.44 (30-min), and 0.94 and 0.34 (24-h), respectively. Employing the resultant baseline narcosis models, toxicity enhancement (Te) values were derived as a ratio of narcosis-predicted over experimental EC50 for the epoxides. For seven aliphatic epoxides, log Te was below 1 in both assays, indicating narcosis-range toxicity with regard to 30-min bioluminescence and 24-h growth inhibition. Concerning eight nonaliphatic epoxides, log Te values up to 2.4 were observed, reflecting excess toxicity through an enhanced electrophilic reactivity of the compounds. Here, however, the intercorrelation between both assays was very low (r2 = 0.09). The results are discussed in terms of electronic substituent effects activating an SN2-type epoxide reaction with nucleophilic protein sites and side-chain activation offering alternative electrophile-nucleophile reaction routes at side-chain sites, leading to respective structural alerts as indicators of excess toxicity. Surprisingly, 30-min bioluminescence appears to be slightly more sensitive to chemical stress than 24-h growth, which holds both for baseline narcotics and for most of the epoxides. This is also reflected by effective narcosis doses 50%, ED50, of 7.1 mmol/kg (30-min) and 7.7 mmol/kg (24-h) estimated from narcosis theory. Keeping in mind the different end points (bioluminescence vs growth) involved, this finding demonstrates that chronic toxicity is not always more sensitive than acute toxicity, calling for analyses with regard to further respective

  13. Patulin biosynthesis: Epoxidation of toluquinol and gentisyl alcohol by particulate preparations from Penicillium patulum

    SciTech Connect

    Priest, J.W.; Light, R.J. )

    1989-11-14

    A crude extract that catalyzes the epoxidation of toluquinol and gentisyl alcohol was isolated from cultures of Penicillium patulum. About 60% of the activity sedimented from crude extract upon centrifugation at 105000g for 2 h, and at 30000g for 30 min after precipitation with 30% ammonium sulfate and resuspension in buffer. The quinone epoxide phyllostine, a product of gentisyl alcohol epoxidation, has previously been shown to be an intermediate in the biosynthesis of patulin and was shown to be further converted to neopatulin by the extract. The epoxide product of toluquinol, desoxyphyllostine (2-methyl-5,6-epoxy-1,4-benzoquinone), has not been reported previously from fungal cultures. Its structure was confirmed by GC-mass spectrometry and proton and {sup 13}C NMR. Its CD spectrum showed the same shape and signs as that of phyllostine, indicating that it too is an enzymatic product with a similar absolute configuration. Whereas chemical epoxidation of toluquinone and gentisly quinone occurs with hydrogen peroxide, the enzymatic epoxidation utilized oxygen and the hydroquinone. The epoxidation was inhibited by 1,10-phenanthroline, EDTA, and {rho}-(chloromercuri)benzenesulfonic acid and by degassing with nitrogen, but no inhibition was observed with KCN, catalase, or CO. The apparent K{sub m}'s were similar for the two substrates with both substrates showing inhibition at 1.0 mM. The rate of desoxyphyllostine formation was more than 10 times that of phyllostine formation at equivalent substrate concentrations. Gentisaldehyde was not a substrate for the enzyme. The epoxidase was induced in late fermentor cultures of P. patulum with the same kinetics as m-hydroxybenzyl alcohol dehydrogenase, another enzyme associated with the induction of patulin biosynthesis.

  14. Organocatalyzed enantioselective desymmetrization of aziridines and epoxides

    PubMed Central

    2013-01-01

    Summary Enantioselective desymmetrization of meso-aziridines and meso-epoxides with various nucleophiles by organocatalysis has emerged as a cutting-edge approach in recent years. This review summarizes the origin and recent developments of enantioselective desymmetrization of meso-aziridines and meso-epoxides in the presence of organocatalysts. PMID:24062828

  15. Poly(monothiocarbonate)s from the Alternating and Regioselective Copolymerization of Carbonyl Sulfide with Epoxides.

    PubMed

    Luo, Ming; Zhang, Xing-Hong; Darensbourg, Donald J

    2016-10-18

    Carbonyl sulfide (COS) is an air pollutant that causes acid rain, ozonosphere damage, and carbon dioxide (CO2) generation. It is a heterocumulene and structural analogue of CO2. Relevant to organic synthesis, it is a source of C═O or C═S groups and thus an ideal one-carbon (C1) building block for synthesizing sulfur-containing polymers through the similar route of CO2 copolymerization. In contrast, traditional synthesis of sulfur-containing polymers often involves the condensation of thiols with phosgene and ring-opening polymerization of cyclic thiocarbonates that are generally derived from thiols and phosgene; thus, COS/epoxide copolymerization is a "greener" route to supplement or supplant current processes for the production of sulfur-containing polymers. This Accounts highlights our efforts on the discovery of the selective formation of poly(monothiocarbonate)s from COS with epoxides via heterogeneous zinc-cobalt double metal cyanide complex (Zn-Co(III) DMCC) and homogeneous (salen)CrX complexes. The catalytic activity and selectivity of Zn-Co(III) DMCC for COS/epoxide copolymerization are similar to those for CO2/epoxide copolymerization. (salen)CrX complexes accompanied by onium salts exhibited high activity and selectivity for COS/epoxide copolymerization under mild conditions, affording copolymers with >99% monothiocarbonate units and high tail-to-head content up to 99%. By way of contrast, these catalysts often show moderate or low activity for CO2/epoxide copolymerization. Of note, a specialty of COS/epoxide copolymerization is the occurrence of an oxygen-sulfur exchange reaction (O/S ER), which may produce carbonate and dithiocarbonate units. O/S ER, which are induced by the metal-OH bond regenerated by chain transfer reactions, can be kinetically inhibited by changing the reaction conditions. We provide a thorough mechanistic understanding of the electronic/steric effect of the catalysts on the regioselectivity of COS copolymerization. The

  16. Physarum polymalic acid hydrolase: Recombinant expression and enzyme activation.

    PubMed

    Mueller, Wolfgang; Haindl, Markus; Holler, Eggehard

    2008-12-19

    As a platform for syntheses of nanoconjugates in antitumor drug delivery, polymalic acid together with its tailoring specific exohydrolase is purified from plasmodium cultures of the slime mold Physarum polycephalum, a member of the phylum myxomycota. Polymalic acid hydrolase is expressed in an inactive form that functions as a molecular adapter for polymalic acid trafficking within the plasmodium and is activated only during secretion. Activation follows specific protein tyrosine phosphorylation and dissociation from plasma membranes. Purified inactive Physarum polymalic acid hydrolase, recombinantly expressed in yeast Saccharomyces, is activated on a preparative basis by the addition of plasma membrane fragments from plasmodia of P. polycephalum. Activation of polymalic acid hydrolase and inhibition of polymalic acid synthesis by protein tyrosine phosphorylation are complementary events and could indicate a joint signal response to plasma membrane damage.

  17. Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens*

    PubMed Central

    Jongkees, Seino A. K.; Yoo, Hayoung; Withers, Stephen G.

    2014-01-01

    Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct 1H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2. PMID:24573682

  18. Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

    PubMed Central

    Stahl, U.; Banas, A.; Stymne, S.

    1995-01-01

    Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

  19. Starch hydrolase inhibitors from edible plants.

    PubMed

    Wang, Hongyu; Liu, Tingting; Huang, Dejian

    2013-01-01

    Type 2 diabetes is a chronic disease with gradual deterioration in glucose metabolism, which causes multiple systemic complications. Postprandial hyperglycemia is a concern in the management of type 2 diabetes. Of all the available antidiabetic therapeutic methods, inhibition of α-glucosidase and α-amylase is postulated to be a preventive treatment. Many natural products and herbal medicines have been recommended as being beneficial for mitigation of postprandial hyperglycemia. In this review, recent discoveries of α-glucosidase and α-amylase inhibitors from edible plants are described along with their chemical structures. Their inhibition mechanisms, the type of each glucosidase and amylase, and measurement methods for the inhibitory activity are also given. Finally, recent progress on low glycemic index foods incorporated with plants containing starch hydrolase inhibitors is summarized. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Polyglycine hydrolases secreted by pathogenic fungi

    USDA-ARS?s Scientific Manuscript database

    Pathogens are known to produce proteases that target host defense proteins. Here we describe polyglycine hydrolases, fungal proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine interdomain linker of targeted plant defense chitinases. Polyglycine hydrolases were puri...

  1. Toluene Monooxygenase-Catalyzed Epoxidation of Alkenes

    PubMed Central

    McClay, Kevin; Fox, Brian G.; Steffan, Robert J.

    2000-01-01

    Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0.01 to 0.33 μmol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified. PMID:10788354

  2. Repurposing Suzuki Coupling Reagents as a Directed Fragment Library Targeting Serine Hydrolases and Related Enzymes

    PubMed Central

    2017-01-01

    Serine hydrolases are susceptible to potent reversible inhibition by boronic acids. Large collections of chemically diverse boronic acid fragments are commercially available because of their utility in coupling chemistry. We repurposed the approximately 650 boronic acid reagents in our collection as a directed fragment library targeting serine hydrolases and related enzymes. Highly efficient hits (LE > 0.6) often result. The utility of the approach is illustrated with the results against autotaxin, a phospholipase implicated in cardiovascular disease. PMID:28564542

  3. Divergent Reactivity via Cobalt Catalysis: An Epoxide Olefination.

    PubMed

    Jamieson, Megan L; Hume, Paul A; Furkert, Daniel P; Brimble, Margaret A

    2016-02-05

    Cobalt salts exert an unexpected and profound influence on the reactivity of epoxides with dimethylsulfoxonium methylide. In the presence of a cobalt catalyst, conditions for epoxide to an oxetane ring expansion instead deliver homoallylic alcohol products, corresponding to a two-carbon epoxide homologation/ring-opening tandem process. The observed reactivity change appears to be specifically due to cobalt salts and is broadly applicable to a variety of epoxides, retaining the initial stereochemistry. This transformation also provides operationally simple access to enantiopure homoallylic alcohols from chiral epoxides without use of organometallic reagents. Tandem epoxidation-homologation of aldehydes in a single step is also demonstrated.

  4. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed

    Mulbry, W W; Karns, J S

    1989-02-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies.

  5. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies. Images PMID:2541658

  6. In Vivo Anti-Tumor Activity and Toxicological Evaluations of Perillaldehyde 8,9-Epoxide, a Derivative of Perillyl Alcohol

    PubMed Central

    Andrade, Luciana Nalone; Amaral, Ricardo Guimarães; Dória, Grace Anne Azevedo; Fonseca, Cecília Santos; da Silva, Tayane Kayane Mariano; Albuquerque Júnior, Ricardo Luiz Cavalcante; Thomazzi, Sara Maria; do Nascimento, Lázaro Gomes; Carvalho, Adriana Andrade; de Sousa, Damião Pergentino

    2016-01-01

    Recent studies have revealed the high cytotoxicity of p-menthane derivatives against human tumor cells. In this study, the substance perillaldehyde 8,9-epoxide, a p-menthane class derivative obtained from (S)-(−)-perillyl alcohol, was selected in order to assess antitumor activity against experimental sarcoma 180 tumors. Toxicological effects related to the liver, spleen, kidneys and hematology were evaluated in mice submitted to treatment. The tumor growth inhibition rate was 38.4%, 58.7%, 35.3%, 45.4% and 68.1% at doses of 100 and 200 mg/kg/day for perillaldehyde 8,9-epoxide, perillyl alcohol and 25 mg/kg/day for 5-FU intraperitoneal treatments, respectively. No toxicologically significant effect was found in liver and kidney parameters analyzed in Sarcoma 180-inoculated mice treated with perillaldehyde 8,9-epoxide. Histopathological analyses of the liver, spleen, and kidneys were free from any morphological changes in the organs of the animals treated with perillaldehyde 8,9-epoxide. In conclusion, the data suggest that perillaldehyde 8,9-epoxide possesses significant antitumor activity without systemic toxicity for the tested parameters. By comparison, there was no statistical difference for the antitumor activity between perillaldehyde 8,9-epoxide and perillyl alcohol. PMID:26742032

  7. Variation in the human soluble epoxide hydrolase gene and risk of restenosis after percutaneous coronary intervention

    PubMed Central

    Kullmann, Silke; Binner, Priska; Rackebrandt, Kirsten; Huge, Andreas; Haltern, Georg; Lankisch, Mark; Füth, Reiner; von Hodenberg, Eberhard; Bestehorn, Hans-Peter; Scheffold, Thomas

    2009-01-01

    Background Restenosis represents the major limiting factor for the long-term efficacy of percutaneous coronary intervention (PCI). Several genetic factors involved in the regulation of the vascular system have been described to play a role in the pathogenesis of restenosis. We investigated whether the EPHX2 K55R polymorphism, previously linked to significantly higher risk for coronary heart disease (CHD), was associated with the occurrence of restenosis after PCI. The association with incident CHD should have been confirmed and a potential correlation of the EPHX2 K55R variant to an increased risk of hypertension was analysed. Methods An overall cohort of 706 patients was studied: This cohort comprised of 435 CHD patients who had undergone successful PCI. Follow-up coronary angiography in all patients was performed 6 months after intervention. Another 271 patients in whom CHD had been excluded by coronary angiography served as controls. From each patient EDTA-blood was drawn at the baseline ward round. Genomic DNA was extracted from these samples and genotyping was performed by real-time PCR and subsequent melting curve analysis. Results In CHD patients 6 month follow-up coronary angiography revealed a restenosis rate of 29.4%, classified as late lumen loss as well as lumen re-narrowing ≥ 50%. Statistical analysis showed an equal genotype distribution in restenosis patients and non-restenosis patients (A/A 82.0% and A/G + G/G 18.0% versus A/A 82.1% and A/G + G/G 17.9%). Moreover, neither a significant difference in the genotype distribution of CHD patients and controls nor an association with increased risk of hypertension was found. Conclusion The results of the present study indicate that the EPHX2 K55R polymorphism is not associated with restenosis after PCI, with incidence of CHD, or with an increased risk of hypertension and therefore, can not serve as a predictor for risk of CHD or restenosis after PCI. PMID:19814804

  8. Variation in the human soluble epoxide hydrolase gene and risk of restenosis after percutaneous coronary intervention.

    PubMed

    Kullmann, Silke; Binner, Priska; Rackebrandt, Kirsten; Huge, Andreas; Haltern, Georg; Lankisch, Mark; Füth, Reiner; von Hodenberg, Eberhard; Bestehorn, Hans-Peter; Scheffold, Thomas

    2009-10-08

    Restenosis represents the major limiting factor for the long-term efficacy of percutaneous coronary intervention (PCI). Several genetic factors involved in the regulation of the vascular system have been described to play a role in the pathogenesis of restenosis. We investigated whether the EPHX2 K55R polymorphism, previously linked to significantly higher risk for coronary heart disease (CHD), was associated with the occurrence of restenosis after PCI. The association with incident CHD should have been confirmed and a potential correlation of the EPHX2 K55R variant to an increased risk of hypertension was analysed. An overall cohort of 706 patients was studied: This cohort comprised of 435 CHD patients who had undergone successful PCI. Follow-up coronary angiography in all patients was performed 6 months after intervention. Another 271 patients in whom CHD had been excluded by coronary angiography served as controls. From each patient EDTA-blood was drawn at the baseline ward round. Genomic DNA was extracted from these samples and genotyping was performed by real-time PCR and subsequent melting curve analysis. In CHD patients 6 month follow-up coronary angiography revealed a restenosis rate of 29.4%, classified as late lumen loss as well as lumen re-narrowing >or= 50%.Statistical analysis showed an equal genotype distribution in restenosis patients and non-restenosis patients (A/A 82.0% and A/G + G/G 18.0% versus A/A 82.1% and A/G + G/G 17.9%). Moreover, neither a significant difference in the genotype distribution of CHD patients and controls nor an association with increased risk of hypertension was found. The results of the present study indicate that the EPHX2 K55R polymorphism is not associated with restenosis after PCI, with incidence of CHD, or with an increased risk of hypertension and therefore, can not serve as a predictor for risk of CHD or restenosis after PCI.

  9. EPOXIDE HYDROLASE ACTIVITIES IN THE MICROSOMES AND THE SOLUBLE FRACTION FROM VICIA SATIVA SEEDLINGS. (R825433)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  10. MICROSOMAL EPOXIDE HYDROLASE (EPHX) POLYMORPHISM AND RISK OF SPONTANEOUS ABORTION. (R825818)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  11. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    SciTech Connect

    Duan, Hongying; Takagi, Akira; Kayano, Hidekazu; Koyama, Isamu; Morisseau, Christophe; Hammock, Bruce D.; Akatsuka, Toshitaka

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell surface has great variation between the cells.

  12. POTENT UREA AND CARBAMATE INHIBITORS OF SOLUBLE EPOXIDE HYDROLASES. (R825433)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  13. POTENT UREA AND CARBAMATE INHIBITORS OF SOLUBLE EPOXIDE HYDROLASES. (R825433)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  14. MICROSOMAL EPOXIDE HYDROLASE (EPHX) POLYMORPHISM AND RISK OF SPONTANEOUS ABORTION. (R825818)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  15. 40 CFR 721.10233 - Linear alkyl epoxide (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Linear alkyl epoxide (generic). 721... Substances § 721.10233 Linear alkyl epoxide (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as linear alkyl epoxide (PMN...

  16. 40 CFR 721.10233 - Linear alkyl epoxide (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Linear alkyl epoxide (generic). 721... Substances § 721.10233 Linear alkyl epoxide (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as linear alkyl epoxide (PMN...

  17. 40 CFR 721.10233 - Linear alkyl epoxide (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Linear alkyl epoxide (generic). 721... Substances § 721.10233 Linear alkyl epoxide (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as linear alkyl epoxide (PMN...

  18. 40 CFR 721.2675 - Perfluoroalkyl epoxide (generic name).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Perfluoroalkyl epoxide (generic name... Substances § 721.2675 Perfluoroalkyl epoxide (generic name). (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as perfluoroalkyl epoxide (PMN P...

  19. 40 CFR 721.2675 - Perfluoroalkyl epoxide (generic name).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Perfluoroalkyl epoxide (generic name... Substances § 721.2675 Perfluoroalkyl epoxide (generic name). (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as perfluoroalkyl epoxide (PMN P...

  20. 40 CFR 721.2675 - Perfluoroalkyl epoxide (generic name).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Perfluoroalkyl epoxide (generic name... Substances § 721.2675 Perfluoroalkyl epoxide (generic name). (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as perfluoroalkyl epoxide (PMN P...

  1. 40 CFR 721.2675 - Perfluoroalkyl epoxide (generic name).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Perfluoroalkyl epoxide (generic name... Substances § 721.2675 Perfluoroalkyl epoxide (generic name). (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as perfluoroalkyl epoxide (PMN P...

  2. 40 CFR 721.2675 - Perfluoroalkyl epoxide (generic name).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Perfluoroalkyl epoxide (generic name... Substances § 721.2675 Perfluoroalkyl epoxide (generic name). (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as perfluoroalkyl epoxide (PMN P...

  3. 21 CFR 172.723 - Epoxidized soybean oil.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Epoxidized soybean oil. 172.723 Section 172.723... CONSUMPTION Other Specific Usage Additives § 172.723 Epoxidized soybean oil. Epoxidized soybean oil may be... reacting soybean oil in toluene with hydrogen peroxide and formic acid. (b) It meets the following...

  4. 21 CFR 172.723 - Epoxidized soybean oil.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Epoxidized soybean oil. 172.723 Section 172.723....723 Epoxidized soybean oil. Epoxidized soybean oil may be safely used in accordance with the following prescribed conditions: (a) The additive is prepared by reacting soybean oil in toluene with hydrogen peroxide...

  5. 21 CFR 172.723 - Epoxidized soybean oil.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Epoxidized soybean oil. 172.723 Section 172.723... CONSUMPTION Other Specific Usage Additives § 172.723 Epoxidized soybean oil. Epoxidized soybean oil may be... reacting soybean oil in toluene with hydrogen peroxide and formic acid. (b) It meets the following...

  6. 21 CFR 172.723 - Epoxidized soybean oil.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Epoxidized soybean oil. 172.723 Section 172.723... CONSUMPTION Other Specific Usage Additives § 172.723 Epoxidized soybean oil. Epoxidized soybean oil may be... reacting soybean oil in toluene with hydrogen peroxide and formic acid. (b) It meets the following...

  7. 21 CFR 172.723 - Epoxidized soybean oil.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Epoxidized soybean oil. 172.723 Section 172.723... CONSUMPTION Other Specific Usage Additives § 172.723 Epoxidized soybean oil. Epoxidized soybean oil may be... reacting soybean oil in toluene with hydrogen peroxide and formic acid. (b) It meets the following...

  8. 40 CFR 721.2673 - Aromatic epoxide resin (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Aromatic epoxide resin (generic). 721... Substances § 721.2673 Aromatic epoxide resin (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as aromatic epoxide resin (PMN P...

  9. 40 CFR 721.2673 - Aromatic epoxide resin (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Aromatic epoxide resin (generic). 721... Substances § 721.2673 Aromatic epoxide resin (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as aromatic epoxide resin (PMN P...

  10. Heterogeneous reactions of epoxides in acidic media.

    PubMed

    Lal, Vinita; Khalizov, Alexei F; Lin, Yun; Galvan, Maria D; Connell, Brian T; Zhang, Renyi

    2012-06-21

    Epoxides have recently been identified as important intermediates in the gas phase oxidation of hydrocarbons, and their hydrolysis products have been observed in ambient aerosols. To evaluate the role of epoxides in the formation of secondary organic aerosols (SOA), the kinetics and mechanism of heterogeneous reactions of two model epoxides, isoprene oxide and α-pinene oxide, with sulfuric acid, ammonium bisulfate, and ammonium sulfate have been investigated using complementary experimental techniques. Kinetic experiments using a fast flow reactor coupled to an ion drift-chemical ionization mass spectrometer (ID-CIMS) show a fast irreversible loss of the epoxides with the uptake coefficients (γ) of (1.7 ± 0.1) × 10(-2) and (4.6 ± 0.3) × 10(-2) for isoprene oxide and α-pinene oxide, respectively, for 90 wt % H(2)SO(4) and at room temperature. Experiments using attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) reveal that diols are the major products in ammonium bisulfate and dilute H(2)SO(4) (<25 wt %) solutions for both epoxides. In concentrated H(2)SO(4) (>65 wt %), acetals are formed from isoprene oxide, whereas organosulfates are produced from α-pinene oxide. The reaction of the epoxides with ammonium sulfate is slow and no products are observed. The epoxide reactions using bulk samples and Nuclear Magnetic Resonance (NMR) spectroscopy reveal the presence of diols as the major products for isoprene oxide, accompanied by aldehyde formation. For α-pinene oxide, organosulfate formation is observed with a yield increasing with the acidity. Large yields of organosulfates in all NMR experiments with α-pinene oxide are attributed to the kinetic isotope effect (KIE) from the use of deuterated sulfuric acid and water. Our results suggest that acid-catalyzed hydrolysis of epoxides results in the formation of a wide range of products, and some of the products have low volatility and contribute to SOA growth under ambient conditions

  11. Cloning and expression of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L.

    PubMed Central

    Maksel, D; Guranowski, A; Ilgoutz, S C; Moir, A; Blackburn, M G; Gayler, K R

    1998-01-01

    The first isolation, cloning and expression of cDNA encoding an asymmetric diadenosine 5',5'''P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) from a higher plant is described. Ap4A hydrolase protein was purified from seeds of both Lupinus luteus and Lupinus angustifolius and partially sequenced. The Ap4A hydrolase cDNA was cloned from L. angustifolius cotyledonary polyadenylated RNA using reverse transcription and PCR with primers based on the amino acid sequence. The cDNA encoded a protein of 199 amino acids, molecular mass 22982Da. When expressed in Escherichia coli fused to a maltose-binding protein, the enzyme catalysed asymmetric cleavage of Ap4A to AMP and ATP which was inhibited at concentrations of F- as low as 3 microM. These are properties characteristic of Ap4A hydrolase (asymmetrical) (EC 3.6.1. 17). Comparison of the Ap4A hydrolase sequences derived from the four known cDNAs from pig, human, lupin and fission yeast showed that, like the mammalian hydrolase, the lupin enzyme possesses a Mut T motif but no other significant similarities. No sequence similarity to the human fragile histidine triad protein, as found in the Ap4A hydrolase from Schizosaccharomyces pombe, was detected in the Ap4A hydrolase from lupin. PMID:9425114

  12. Alkene epoxidation employing metal nitro complexes

    DOEpatents

    Andrews, M.A.; Cheng, C.W.; Kelley, K.P.

    1982-07-15

    Process for converting alkenes to form epoxides utilizes transition metal nitro complexes of the formula: M(RCN)/sub 2/XNO/sub 2/ wherein M is palladium or platinum, R is an alkyl or aryl group containing up to 12 carbon atoms, and X is a monoanionic, monodentate ligand such as chlorine, optionally in the presence of molecular oxygen.

  13. Epoxidation of Methyl Oleate using Heterogeneous Catalyst

    USDA-ARS?s Scientific Manuscript database

    In this work we studied the catalytic activity of commercial alumina, and laboratory synthesized alumina doped with Lewis acid metals, in the epoxidation of methyl oleate with aqueous hydrogen peroxide. It was observed that the reaction yields increased when the amount of catalyst, the quantity of ...

  14. Polymerization of epoxidized triglycerides with fluorosulfonic acid

    USDA-ARS?s Scientific Manuscript database

    The use of triglycerides as agri-based renewable raw materials for the development of new products is highly desirable in view of uncertain future petroleum prices. A new method of polymerizing epoxidized soybean oil has been devised with the use of fluorosulfonic acid. Depending on the reaction con...

  15. Epoxide-mediated CifR repression of cif gene expression utilizes two binding sites in Pseudomonas aeruginosa.

    PubMed

    Ballok, Alicia E; Bahl, Christopher D; Dolben, Emily L; Lindsay, Allia K; St Laurent, Jessica D; Hogan, Deborah A; Madden, Dean R; O'Toole, George A

    2012-10-01

    Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees.

  16. Epoxide-Mediated CifR Repression of cif Gene Expression Utilizes Two Binding Sites in Pseudomonas aeruginosa

    PubMed Central

    Ballok, Alicia E.; Bahl, Christopher D.; Dolben, Emily L.; Lindsay, Allia K.; St. Laurent, Jessica D.; Hogan, Deborah A.; Madden, Dean R.

    2012-01-01

    Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the −10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees. PMID:22843844

  17. Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

    PubMed Central

    Parkkari, Teija; Haavikko, Raisa; Laitinen, Tuomo; Navia-Paldanius, Dina; Rytilahti, Roosa; Vaara, Miia; Lehtonen, Marko; Alakurtti, Sami; Yli-Kauhaluoma, Jari; Nevalainen, Tapio; Savinainen, Juha R.; Laitinen, Jarmo T.

    2014-01-01

    Background α/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design. Methodology/Principal Findings Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors. Conclusions/Significance We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore

  18. Mechanics of Crack Growth in Epoxide Adhesives

    DTIC Science & Technology

    1978-05-01

    mind when considering the static G (arrest) values shown in Figure 3. Ic 9 First, Bascom et al 7 ’I 0 have elegantly demonstrated that for rubber ...in limited regions of the material. The diameter of the pores or channels in a craze is typically a few tens of nanometres and the void content is...about 40 to 60%. However, while there is definite proof for craze formation in rubber -modified epoxide 24 materials, the evidence for craze formation in

  19. Effects of proteins and polynucleotides on the activity of various hydrolases

    PubMed Central

    Palmieri, M. J.; Koldovský, O.

    1972-01-01

    The effect of various macromolecules on the activity of several hydrolases was studied. Dilution of partially purified acid β-galactosidase from ileal mucosa of suckling rats resulted in a decrease of specific activity. The relationship between specific activity and dilution of the enzyme suggests a dissociation of the enzyme. This could be prevented by addition of several proteins tested. However, addition of DNA to the assay mixture for acid β-galactosidase caused an inhibition. This inhibition could be prevented by addition of proteins. Other polynucleotides and tRNA also exert an inhibitory effect that is prevented by albumin, but nucleotides have no effect. This inhibition occurs maximally at a low pH (3.0–4.0); no inhibition is observed at pH5.5. A similar pH-dependent inhibition by DNA was also found with various other acid hydrolases. PMID:5076227

  20. The electron-impact promoted fragmentation of aurone epoxides.

    NASA Technical Reports Server (NTRS)

    Brady, B. A.; O'Sullivan, W. I.; Duffield, A. M.

    1972-01-01

    The mass spectra of six aurone epoxides have been rationalized with the aid of high resolution mass spectrometry and metastable ion evidence. These compounds fragment in a well defined manner and mechanisms are proposed for the formation of their characteristic ions. Some similarity was observed between the mass spectra of 6-methoxyaurone epoxide (II), 4-hydroxy-7-methoxy-3-phenylcoumarin (VII) and 7-methoxyflavonol (IX). The possibility that VII and IX are intermediates in the fragmentation of epoxide II is discussed. Thermal rearrangement of aurone epoxide II was shown to yield the corresponding flavonol IX and coumarin VII.

  1. Simple Epoxide Formation for the Organic Laboratory Using Oxone

    ERIC Educational Resources Information Center

    Broshears, Williams C.; Esteb, John J.; Richter, Jeremy; Wilson, Anne M.

    2004-01-01

    Epoxide chemistry is widely used in organic synthesis and regularly discussed in organic chemistry textbooks. An experiment to generate dimethyldioxirane in situ from acetone using Oxone is explained.

  2. Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Alapafuja, Shakiru O.; Nikas, Spyros P.; Bharatan, Indu; Shukla, Vidyanand G.; Nasr, Mahmoud L.; Bowman, Anna L.; Zvonok, Nikolai; Li, Jing; Shi, Xiaomeng; Engen, John R.; Makriyannis, Alexandros

    2013-01-01

    Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs. PMID:23083016

  3. Transformation Kinetics of Chlorinated Ethenes by Methylosinus trichosporium OB3b and Detection of Unstable Epoxides by On-Line Gas Chromatography

    PubMed Central

    van Hylckama, Vlie... J.; de Koning, W.; Janssen, D. B.

    1996-01-01

    A rapid and accurate method for the determination of transformation kinetics of volatile organic substrates was developed. Concentrations were monitored by on-line gas chromatographic analysis of the headspace of well-mixed incubation mixtures. With this method, the kinetics of transformation of a number of C(inf1) and C(inf2) halogenated alkanes and alkenes by Methylosinus trichosporium OB3b expressing particulate methane monooxygenase or soluble methane monooxygenase (sMMO) were studied. Apparent specific first-order rate constants for cells expressing sMMO decreased in the order of dichloromethane, vinyl chloride, cis-1,2-dichloroethene, trans-1,2-dichloroethene, 1,1-dichloroethene, trichloroethene, chloroform, and 1,2-dichloroethane. During the degradation of trichloroethene, cis-1,2-dichloroethene, trans-1,2-dichloroethene, and vinyl chloride, the formation of the corresponding epoxides was observed. The epoxide of vinyl chloride and the epoxide of trichloroethene, which temporarily accumulated in the medium, were chemically degraded according to first-order kinetics, with half-lives of 78 and 21 s, respectively. Cells expressing sMMO actively degraded the epoxide of cis-1,2-dichloroethene but not the epoxide of trans-1,2-dichloroethene. Methane and acetylene inhibited degradation of the epoxide of cis-1,2-dichloroethene, indicating that sMMO was involved. PMID:16535402

  4. Phosphonoacetic acid utilization by fungal isolates: occurrence and properties of a phosphonoacetate hydrolase in some penicillia.

    PubMed

    Forlani, Giuseppe; Klimek-Ochab, Magdalena; Jaworski, Jakub; Lejczak, Barbara; Picco, Anna M

    2006-12-01

    Among a collection of 18 fungal strains representing eight genera, only two strains (Penicillium oxalicum and P. minioluteum) were capable of growth on phosphonoacetic acid as sole phosphorous source. Enrichment liquid cultures in minimal medium with the compound as the only P-source selected four isolates, that were also identified as Penicillium spp. Phosphonoacetate metabolism did not lead to extracellular release of inorganic phosphate. In all cases phosphonoacetate hydrolase activity was detected in partially purified extracts, and a protein of the expected molecular mass reacted with polyclonal antibodies raised against the enzyme from P. oxalicum. There was no relation between phosphonoacetate hydrolase specific activity and growth rate or yield. Phosphonoacetic acid was the inducer of the hydrolase, independently of the concurrent availability of inorganic phosphate. Notwithstanding this, the utilization of the phosphonate was significantly inhibited in the presence of phosphate, suggesting an interference of the latter with phosphonoacetic acid uptake.

  5. Murein hydrolase activity of surface layer proteins from Lactobacillus acidophilus against Escherichia coli.

    PubMed

    Meng, Jun; Gao, Shu-Ming; Zhang, Qiu-Xiang; Lu, Rong-Rong

    2015-08-01

    The aim of this study was to investigate the murein hydrolase activities of the surface layer proteins (SLPs) from two strains of Lactobacillus acidophilus using zymography. The influence of these hydrolase activities on Escherichia coli ATCC 43893 was also evaluated by analysing their growth curve, cell morphology and physiological state. After the incubation of E. coli with SLPs, growth was inhibited, the number of viable cells was significantly reduced, examination by transmission electron microscopy showed that the cell wall was damaged and flow cytometry results indicated that the majority of the cells were sublethally injured. All of these results suggested that the SLPs of both L. acidophilus strains possessed murein hydrolase activities that were sublethal to E. coli cells. Copyright © 2015. Published by Elsevier B.V.

  6. Chiral Ketone and Iminium Catalysts for Olefin Epoxidation

    NASA Astrophysics Data System (ADS)

    Wong, O. Andrea; Shi, Yian

    Organo-catalyzed asymmetric epoxidation has received much attention in the past 30 years and significant progress has been made for various types of olefins. This review will cover the advancement made in the field of chiral ketone and chiral iminium salt-catalyzed epoxidations.

  7. Epoxides--is there a human health problem?

    PubMed Central

    Manson, M M

    1980-01-01

    The purpose of this review is to consider whether epoxides represent a hazard to human health. Possible means of occupational and non-occupational exposure are discussed with reference to the production and uses of industrially important compounds and other epoxides, such as naturally occurring plant and fungal products. In addition to epoxides themselves, unsaturated compounds that may be metabolised in vivo to epoxides are included, since this appears to be a further important means of exposure. The toxicology, in particular carcinogenicity and mutagenicity, is discussed, along with a brief outline of the biochemistry such as metabolism, binding to cell constituents, and DNA repair mechanisms. The question of interactions between different epoxides in vivo is also raised. PMID:7004476

  8. Regiospecific Epoxidation of Carvone: A Discovery-Oriented Experiment for Understanding the Selectivity and Mechanism of Epoxidation Reactions

    ERIC Educational Resources Information Center

    Mak, Kendrew K. W.; Lai, Y. M.; Siu, Yuk-Hong

    2006-01-01

    This article describes a discovery-oriented experiment for demonstrating the selectivity of two epoxidation reactions. Peroxy acids and alkaline H[subscript 2]O[subscript 2] are two commonly used reagents for alkene epoxidation. The former react preferentially with electron-rich alkenes while the latter works better with alpha,beta-unsaturated…

  9. Hydrolase-like properties of a cofactor-independent dioxygenase.

    PubMed

    Thierbach, Sven; Büldt-Karentzopoulos, Klaudia; Dreiling, Alena; Hennecke, Ulrich; König, Simone; Fetzner, Susanne

    2012-05-29

    Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an α/β-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the α/β-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.

  10. Uptake kinetics of three epoxides into sulfuric acid solution

    NASA Astrophysics Data System (ADS)

    Wang, Tianhe; Liu, Ze; Wang, Weigang; Ge, Maofa

    2012-09-01

    This work presents a study of the uptake of isoprene epoxide, butadiene epoxide (BMO) and butadiene diepoxide (BDO) into sulfuric acid solutions which helps to understand the reactivity of epoxides existing in the atmosphere toward acidic aerosols. The uptake of these three compounds into 0-30 wt % H2SO4 solutions were measured using a rotated wetted-wall reactor (RWW) coupled to a single-photon ionization time of flight mass spectrometer (SPI-TOFMS). The epoxides were found to be very easily taken up by H2SO4 solutions even in dilute concentrations of pH levels. Isoprene epoxide was found to partition reversibly into solution at pH = 4, whereas irreversible uptake was observed when pH ≤ 3. We reported the reactive uptake coefficients from 1.87 × 10-5 to 2.67 × 10-3 for pH = 3-20 wt % H2SO4 solutions. A chemical reaction for isoprene epoxide was responsible for the reactive uptake. By means of mass spectrometry, gas chromatography and FTIR spectroscopy, a gas product was identified to be 2-methyl-3-butenal. The uptake behavior of butadiene epoxide was similar with that of isoprene epoxide, while butadiene diepoxide partitioned irreversibly over the whole acidity range of 0-30 wt %, and the reactive uptake coefficients increased slightly (0.849 × 10-4-1.36 × 10-4) from pure water to pH = 1. The reactivity that displayed close dependence on the hydrolysis rates of the three epoxides was analyzed and compared according to their molecular structural differences. The atmospheric lifetimes were calculated and atmospheric implication was discussed based on the corresponding reactive uptake coefficients.

  11. 1,2,3-Triazolylalkylribitol derivatives as nucleoside hydrolase inhibitors.

    PubMed

    Goeminne, A; McNaughton, M; Bal, G; Surpateanu, G; Van der Veken, P; De Prol, S; Versées, W; Steyaert, J; Apers, S; Haemers, A; Augustyns, K

    2007-05-01

    A range of novel 1,2,3-triazolylalkylribitol derivatives were synthesized and evaluated as nucleoside hydrolase inhibitors. The most active compound (11a) has low micromolar potency and is structurally diverse from previously reported nucleoside hydrolase inhibitors, which, along with the simplicity of the chemistry involved in its synthesis, makes it a good lead for the further development of novel nucleoside hydrolase inhibitors.

  12. Swelling behaviour in n-pentane and mechanical properties of epoxidized natural rubber with different epoxide content

    NASA Astrophysics Data System (ADS)

    Kinasih, N. A.; Fathurrohman, M. I.; Winarto, D. A.

    2017-07-01

    Epoxidized natural rubber (ENR) with different level of epoxidation (i.e. 10, 20, 30, 40 and 50 mol% indicated as ENR ENR10, ENR20, ENR30, ENR40 and ENR50, respectively) were prepared. They were then vulcanized by using efficient system vulcanization. The effect of epoxide content on curing characteristic, swelling and mechanical properties in N-pentane was investigated. The Attenuated Resonance Fourier Transform Infrared (ATR-FTIR) and H-Nuclear Magnetic Resonance (H-NMR) were used to determine the epoxidation level. Glass transition (Tg) of ENR samples was determined by using Direct Scanning Calorimetry (DSC). The result revealed that the resistance of ENR in N-pentane increased with increasing epoxidation level, which indicated by decreasing equilibrium mol uptake and diffusion coefficient. The compression set of ENR and aging resistance increased with increasing epoxide content, except ENR50 was due to ENR 50 have two Tg value. However, the value of hardness and tensile strength were not effected by epoxidation level.

  13. Carcinogenicity and mechanistic insights on the behavior of epoxides and epoxide-forming chemicals.

    PubMed

    Melnick, Ronald L

    2002-12-01

    Many epoxides and their precursors are high production volume chemicals that have major uses in the polymer industry and as intermediates in the manufacture of other chemicals. Several of these chemicals were demonstrated to be carcinogenic in laboratory animal studies conducted by the Ramazzini Foundation (e.g., vinyl chloride, acrylonitrile, styrene, styrene oxide, and benzene) and by the National Toxicology Program (e.g., ethylene oxide, 1,3-butadiene, isoprene, chloroprene, acrylonitrile, glycidol, and benzene). The most common sites of tumor induction were lung, liver, harderian gland, and circulatory system in mice; Zymbal's gland and brain in rats; and mammary gland and forestomach in both species. Differences in cancer outcome among studies of epoxide chemicals may be related to differences in study design (e.g., dose, duration, and route of exposure; observation period; animal strains), as well as biological factors affecting target organ dosimetry of the DNA-reactive epoxide (toxicokinetics) and tissue response (toxicodynamics). N7-Alkylguanine, N1-alkyladenine, and cyclic etheno adducts, as well as K-ras and p53 mutations, have been detected in animals and/or workers exposed to several of these chemicals. The classifications of these chemical carcinogens by IARC and NTP are based on animal and human data and results of mechanistic studies. Reducing occupational and environmental exposures to these chemicals will certainly reduce human cancer risks.

  14. Terreic Acid, a Quinone Epoxide Inhibitor of Bruton's Tyrosine Kinase

    NASA Astrophysics Data System (ADS)

    Kawakami, Yuko; Hartman, Stephen E.; Kinoshita, Eiji; Suzuki, Hidefumi; Kitaura, Jiro; Yao, Libo; Inagaki, Naoki; Franco, Alessandra; Hata, Daisuke; Maeda-Yamamoto, Mari; Fukamachi, Hiromi; Nagai, Hiroichi; Kawakami, Toshiaki

    1999-03-01

    Bruton's tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors.

  15. Twisting of glycosidic bonds by hydrolases

    USDA-ARS?s Scientific Manuscript database

    Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bond...

  16. Structure and function of polyglycine hydrolases

    USDA-ARS?s Scientific Manuscript database

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave polyglycine linkers of targeted plant defense chitinases. Unlike typical endoproteases that cleave a specific peptide bond, these 640 amino acid glycoproteins selectively cleave one of multiple peptide bonds within polyglyci...

  17. Synthesis and reactivity of coumarin 3,4-epoxide.

    PubMed

    Born, S L; Rodriguez, P A; Eddy, C L; Lehman-McKeeman, L D

    1997-11-01

    Coumarin is used widely as a fragrance constituent and is administered clinically in the treatment of certain lymphedemas and malignancies. Although toxicity occurs only rarely in humans treated clinically with high-dose coumarin, it is well established that coumarin is hepatotoxic in the rat. This species difference in susceptibility to toxicity reflects the disparate metabolic processes occurring in humans and rodents. In humans, coumarin is converted extensively via cytochrome P450 2A6 to the nontoxic 7-hydroxycoumarin metabolite. In contrast, coumarin 3,4-epoxidation is thought to predominate in rodent species, resulting in the formation of several potentially toxic metabolites. Coumarin epoxide is thought to be highly unstable and has not been isolated synthetically or as a microsomal product. To address this issue, coumarin 3,4-epoxide was synthesized, and its stability and fate have been determined. Coumarin 3,4-epoxide was prepared by reacting coumarin with dimethyldioxirane. The epoxide was stable in organic solvents and survived conditions required for analysis by gas chromotography. Its structure was confirmed via 1H-NMR and gas chromatography-mass spectrometry-infrared spectroscopy (GC-MS-IR). In contrast, coumarin 3,4-epoxide was unstable in aqueous solution, converting within 20 sec to a ring-opened compound. Using GC-MS-IR analysis, the single coumarin 3,4-epoxide product was identified as o-hydroxyphenylacetaldehyde (o-HPA). Although other investigators have suggested that 3-hydroxycoumarin is an intermediate in o-HPA formation from coumarin 3,4-epoxide, we have demonstrated that 3-hydroxycoumarin, incubated in an aqueous system or with liver microsomal proteins, does not form o-HPA. Thus, the results of the present work establish that coumarin 3,4-epoxide can be synthesized and that o-HPA, which has previously been shown to be a prominent coumarin metabolite in rat liver microsomal incubations, is formed directly from coumarin 3,4-epoxide. These

  18. Iron-catalyzed olefin epoxidation in the presence of acetic acid: insights into the nature of the metal-based oxidant.

    PubMed

    Mas-Ballesté, Rubén; Que, Lawrence

    2007-12-26

    The iron complexes [(BPMEN)Fe(OTf)2] (1) and [(TPA)Fe(OTf)2] (2) [BPMEN = N,N'-bis-(2-pyridylmethyl)-N,N'-dimethyl-1,2-ethylenediamine; TPA = tris-(2-pyridylmethyl)amine] catalyze the oxidation of olefins by H2O2 to yield epoxides and cis-diols. The addition of acetic acid inhibits olefin cis-dihydroxylation and enhances epoxidation for both 1 and 2. Reactions carried out at 0 degrees C with 0.5 mol % catalyst and a 1:1.5 olefin/H2O2 ratio in a 1:2 CH3CN/CH3COOH solvent mixture result in nearly quantitative conversions of cyclooctene to epoxide within 1 min. The nature of the active species formed in the presence of acetic acid has been probed at low temperature. For 2, in the absence of substrate, [(TPA)FeIII(OOH)(CH3COOH)]2+ and [(TPA)FeIVO(NCCH3)]2+ intermediates can be observed. However, neither is the active epoxidizing species. In fact, [(TPA)FeIVO(NCCH3)]2+ is shown to form in competition with substrate oxidation. Consequently, it is proposed that epoxidation is mediated by [(TPA)FeV(O)(OOCCH3)]2+, generated from O-O bond heterolysis of the [(TPA)FeIII(OOH)(CH3COOH)]2+ intermediate, which is promoted by the protonation of the terminal oxygen atom of the hydroperoxide by the coordinated carboxylic acid.

  19. Synthesis of functionalized epoxides by copper-catalyzed alkylative epoxidation of allylic alcohols with alkyl nitriles.

    PubMed

    Bunescu, Ala; Wang, Qian; Zhu, Jieping

    2015-04-17

    A copper-catalyzed oxyalkylation of allylic alcohols using nonactivated alkyl nitriles as reaction partners was developed. A sequence involving generation of an alkyl nitrile radical followed by its addition to a double bond and a copper-mediated formation of C(sp(3))-O bond was proposed to account for the reaction outcome. The protocol provided an efficient route to functionalized tri- and tetrasubstituted epoxides via formation of a C(sp(3))-C(sp(3)) and a C(sp(3))-O bond with moderate to excellent diastereoselectivity.

  20. Dienelactone hydrolase from Pseudomonas sp. strain B13.

    PubMed Central

    Ngai, K L; Schlömann, M; Knackmuss, H J; Ornston, L N

    1987-01-01

    Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13. PMID:3804973

  1. Bacterial Cyanuric Acid Hydrolase for Water Treatment.

    PubMed

    Yeom, Sujin; Mutlu, Baris R; Aksan, Alptekin; Wackett, Lawrence P

    2015-10-01

    Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 μM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 μM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Bacterial Cyanuric Acid Hydrolase for Water Treatment

    PubMed Central

    Yeom, Sujin; Mutlu, Baris R.; Aksan, Alptekin

    2015-01-01

    Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 μM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 μM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation. PMID:26187963

  3. Product formation controlled by substrate dynamics in leukotriene A4 hydrolase.

    PubMed

    Stsiapanava, Alena; Tholander, Fredrik; Kumar, Ramakrishnan B; Qureshi, Abdul Aziz; Niegowski, Damian; Hasan, Mahmudul; Thunnissen, Marjolein; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

    2014-02-01

    Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers.

  4. Presence of epoxide hydrolase activity in Aspergillus niger: Hydrolysis of 6', 7'-epoxybergamottin to 6', 7'-dihydroxybergamottin

    USDA-ARS?s Scientific Manuscript database

    The 6', 7'-epoxybergamottin (EB) is one of major furanocoumarins in grapefruit. Previously, we have shown that Aspergillus niger has a capability of metabolizing EB into 6', 7'-dihydroxybergamottin (DHB), which is further metabolized to bergaptol and bergaptol-5-sulfate in vivo. In this study, we at...

  5. [Effect of genetic polymorphisms of microsomal epoxide hydrolase on urinary 1-hydroxypyrene levels in coke oven workers].

    PubMed

    Leng, Shu-Guang; Zheng, Yu-Xin; Huang, Chuan-Feng; Dai, Yu-Fei; Li, Xiao-Hua; Niu, Yong; Pan, Zu-Fei; Li, Tao; He, Feng-Sheng

    2004-08-01

    To investigate the associations of polymorphisms of metabolic enzyme genes with urinary 1-hydroxypyrene levels in coke oven workers. One hundred and forty-eight workers from a coke oven plant and 69 controls without occupational PAHs exposure were selected in this study. Urinary 1-hydroxypyrene was detected by high performance liquid chromatography with florescence detector. The genotypes at I462V site in exon 7 of CYP1A1 gene, GSTM1, GSTT1, I105V site in GSTP1gene, Pst1 and Dra1 sites in CYP2E1 gene, P187S site in NQO1 gene, Kpn1, BamH1 and Taq1 sites in NAT2 gene, and H113Y, R139H sites in mEH gene were determined by PCR-based methods. Personal information including occupational exposure history, age, sex, smoking and drinking status was collected by the questionnaire. The level of urinary 1-hydroxypyrene in coke oven workers [(5.61 +/- 1.04) mol/mol Cr] was higher than that in control [(0.74 +/- 0.32) micro mol/mol Cr]. After adjusting external occupational exposure category and smoking, coke oven workers with variant homozygotes at H113Y site of mEH gene had significantly higher urinary 1-hydroxypyrene concentrations than those with heterozygotes, and wild homozygotes (6.41 +/- 1.09 vs. 6.24 +/- 1.08, and 4.62 +/- 0.95 micro mol/mol Cr, P < 0.05), and gene-gene interaction was found between CYP1A1 and mEH. Genetic polymorphism of mEH gene could be a susceptible biomarker in coke oven workers which was involved in the individual susceptibility on metabolism of PAHs.

  6. Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase

    PubMed Central

    Cadel, Sandrine; Foulon, Thierry; Viron, Annie; Balogh, Agnès; Midol-Monnet, Stéphanie; Noël, Nadine; Cohen, Paul

    1997-01-01

    An aminopeptidase B (Ap-B) was previously purified to homogeneity from rat testis extracts and characterized. In the present work, by using oligonucleotides selected on the basis of partial amino acid microsequences of pure Ap-B and PCR techniques, the nucleotide sequence of a 2.2-kb cDNA was obtained. The deduced amino acid sequence corresponds to a 648-residue protein (72.3 kDa) containing the canonical “HEXXHX18E” signature, which allowed its classification as a member of the M1 family of metallopeptidases. It exhibits 33% identity and 48% similarity with leukotriene-A4 hydrolase, a relation further supported by the capacity of Ap-B to hydrolyze leukotriene A4. Both enzymes also were closely related to a partially sequenced protein from Dictyostelium discoideum, which might constitute the putative common ancestor of either aminopeptidase or epoxide hydrolase, or both. Ap-B and its mRNA were detected in the germ line and in the Sertoli and peritubular cells of the seminiferous tubules. Because the enzyme was found in the medium conditioned by spermatocytes and spermatids and in the acrosome during spermatozoa formation, together these observations suggested an involvement of this exometallopeptidase in the secretory pathway. It is concluded that this ubiquitous enzyme may be involved in multiple processing mechanisms. PMID:9096329

  7. Asymmetric catalysis of epoxide ring-opening reactions.

    PubMed

    Jacobsen, E N

    2000-06-01

    The discovery of the metal salen-catalyzed asymmetric ring-opening (ARO) of epoxides is chronicled. A screening approach was adopted for the identification of catalysts for the addition of TMSN(3) to meso-epoxides, and the chiral (salen)CrN(3) complex was identified as optimal. Kinetic and structural studies served to elucidate the mechanism of catalysis, which involves cooperative activation of both epoxide and azide by two different metal centers. Covalently linked bimetallic complexes were constructed on the basis of this insight, and shown to catalyze the ARO with identical enantioselectivity but 1-2 orders of magnitude greater reactivity than the monomeric analogues. Extraordinarily high selectivity is observed in the kinetic resolution of terminal epoxides using the (salen)CrN(3)/TMSN(3) system. A search for a practical method for the kinetic resolution reaction led to the discovery of highly enantiomer-selective hydrolytic ring-opening using the corresponding (salen)Co(III) catalyst. This system displays extraordinary substrate generality, and allows practical access to enantiopure terminal epoxides on both laboratory and industrial scales.

  8. Autonomic self-healing in epoxidized natural rubber.

    PubMed

    Rahman, Arifur; Sartore, Luciana; Bignotti, Fabio; Di Landro, Luca

    2013-02-01

    The development of polymers that can repair damage autonomously would be useful to improve the lifetime of polymeric materials. To date, limited attention has been dedicated to developing elastomers with autonomic self-healing ability, which can recover damages without need for an external or internal source of healing agents. This work investigates the self-healing behavior of epoxidized natural rubber (ENR) with two different epoxidation levels (25 and 50 mol % epoxidation) and of the corresponding unfunctionalized rubber, cis-1,4-polyisoprene (PISP). A self-adhesion assisted self-healing behavior was revealed by T-peel tests on slightly vulcanized rubbers. A higher epoxidation level was found to enhance self-healing. Self-healing of rubbers following ballistic damages was also investigated. A pressurized air flow test setup was used to evaluate the self-healing of ballistic damages in rubbers. Microscope (OM, SEM, and TEM) analyses were carried out to provide further evidence of healing in the impact zones. Self-healing of ballistic damages was observed only in ENR with 50 mol % epoxidation and it was found to be influenced significantly by the cross-link density. Finally, self-healing of ballistic damages was also observed in ENR50/PISP blends only when the content of the healing component (i.e., ENR50) was at least 25 wt %. From an analysis of the results, it was concluded that a synergistic effect between interdiffusion and interaction among polar groups leads to self-healing in ENR.

  9. Partial purification and characterization of an inducible indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans

    SciTech Connect

    Chou, Jyh-Ching |; Cohen, J.D.; Mulbry, W.W.

    1996-11-01

    Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH{sub 4}){sub 2}SO{sub 4}, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg{sup 2+} the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO{sub 4}; the activity was increased by 40% with 1mM MnSO{sub 4}. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 45 refs., 5 figs., 3 tabs.

  10. Characterization of tunable piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases

    PubMed Central

    Long, Jonathan Z.; Jin, Xin; Adibekian, Alexander; Li, Weiwei; Cravatt, Benjamin F.

    2010-01-01

    Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the serine hydrolase superfamily that degrade the endocannabinoids 2-arachidonoylglycerol and anandamide, respectively. We have recently discovered that MAGL and FAAH are both inhibited by carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous functions of MAGL and FAAH. Herein, we disclose a full account of the syntheses, structure-activity relationships, and inhibitory activities of piperidine/piperazine carbamates against members of the serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH inhibition by the attachment of an appropriately substituted bisarylcarbinol or aryloxybenzyl moiety, respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated inhibitory activity, suggesting a strict requirement for a six-member ring to maintain potency. PMID:20099888

  11. Ubiquitin dimers control the hydrolase activity of UCH-L3.

    PubMed

    Setsuie, Rieko; Sakurai, Mikako; Sakaguchi, Yuriko; Wada, Keiji

    2009-01-01

    Ubiquitin (Ub) carboxy terminal hydrolase (UCH)-L1 and UCH-L3 are two of the deubiquitinating enzymes expressed in the brain. Both gad mice, which lack UCH-L1 expression and Uchl3 knockout mice exhibit neurodegeneration, although at distinct areas. These phenotypes indicate the importance of UCH-L1 and UCH-L3 in the regulation of the central nervous system. However, molecular substrates and the molecular regulators of UCH-L1 and UCH-L3 remain poorly identified. Here we show that Ub dimers interact non-covalently with UCH-L3 in vitro and in cells. These interactions were not observed with UCH-L1 in cells. In vitro, K48-linked Ub dimers pronouncedly inhibited the hydrolase activity of UCH-L3, while mono-Ub, a previously identified interacting protein, inhibited the hydrolase activity of UCH-L1. These results indicate that mono-Ub and Ub dimers may regulate the enzymatic functions of UCH-L1 and UCH-L3, respectively, in vivo.

  12. Sulfuric acid as a catalyst for ring-opening of biobased bis-epoxides

    USDA-ARS?s Scientific Manuscript database

    Vegetable oils can be relatively and easily transformed into bio-based epoxides. Because of this, the acid-catalyzed epoxide ring-opening has been explored for the preparation of bio-based lubricants and polymers. Detailed model studies are carried out only with mono-epoxide made from methyl oleate,...

  13. Formation of furan fatty alkyl esters from their bis-epoxide fatty esters

    USDA-ARS?s Scientific Manuscript database

    Epoxidation of vegetable oils and consecutive epoxide ring-opening reaction is a widely investigated path for producing biobased lubricants and polymers. The reaction mechanism and products are considered well-studied and known. In the current study, the reactions of epoxidized alkyl soyate with fou...

  14. Lithium BINOL Phosphate Catalyzed Desymmetrization of meso-Epoxides with Aromatic Thiols

    PubMed Central

    2015-01-01

    A highly enantioselective method for desymmetrization of meso-epoxides using thiols is reported. This is the first example of epoxide activation achieved using metal BINOL phosphates. The reaction has a broad scope in terms of epoxide substrates and aromatic thiol nucleophiles. The resulting β-hydroxyl sulfides are obtained in excellent yield and enantioselectivity. PMID:25317934

  15. 40 CFR 721.10210 - Soybean oil, epoxidized, reaction products with diethanolamine.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Soybean oil, epoxidized, reaction... Significant New Uses for Specific Chemical Substances § 721.10210 Soybean oil, epoxidized, reaction products... chemical substance identified as soybean oil, epoxidized, reaction products with diethanolamine (PMN...

  16. 40 CFR 721.10210 - Soybean oil, epoxidized, reaction products with diethanolamine.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Soybean oil, epoxidized, reaction... Significant New Uses for Specific Chemical Substances § 721.10210 Soybean oil, epoxidized, reaction products... chemical substance identified as soybean oil, epoxidized, reaction products with diethanolamine (PMN...

  17. 40 CFR 721.10210 - Soybean oil, epoxidized, reaction products with diethanolamine.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Soybean oil, epoxidized, reaction... Significant New Uses for Specific Chemical Substances § 721.10210 Soybean oil, epoxidized, reaction products... chemical substance identified as soybean oil, epoxidized, reaction products with diethanolamine (PMN...

  18. 40 CFR 721.10210 - Soybean oil, epoxidized, reaction products with diethanolamine.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Soybean oil, epoxidized, reaction... Significant New Uses for Specific Chemical Substances § 721.10210 Soybean oil, epoxidized, reaction products... chemical substance identified as soybean oil, epoxidized, reaction products with diethanolamine (PMN...

  19. Synthesis and Tribological Studies of Branched Alcohol Derived Epoxidized Biodiesel

    PubMed Central

    Ren, Qinggong; Pan, Jingjing; Zhou, Jie; Na, Yinna; Chen, Changle; Li, Weimin

    2015-01-01

    The optimization and kinetics of the ring-opening reaction of an epoxidized biodiesel (epoxidized rapeseed oil methyl ester) (EBD) with 2-ethyl hexanol (2-EH) were studied. The determined optimum conditions were 4:1 2-EH/oil molar ratio, 90 °C, 18 h, and 7 wt % of Amberlyst D001 (dry) catalyst; the product’s oxirane oxygen content was 0.081% with 38.32 mm2/s viscosity at 40 °C. The catalyst retained its high catalytic power after recycling five times. Furthermore, the determined non-catalyzed activation energy was 76 kJ·mol−1 and 54 kJ·mol−1 with the D001 resin catalyst. The product’s chemical structure was investigated through FT-IR and 1H NMR. The viscosity, flash point, pour point, and anti-wear properties of the product were improved compared with those of epoxidized biodiesel. PMID:28793587

  20. Reinforcing polymer composites with epoxide-grafted carbon nanotubes.

    PubMed

    Wang, Shiren; Liang, Richard; Wang, Ben; Zhang, Chuck

    2008-02-27

    An in situ functionalization method was used to graft epoxide onto single-walled carbon nanotubes (SWNTs) and improve the integration of SWNTs into epoxy polymer. The characterization results of Raman, FT-IR and transmission electron microscopy (TEM) validated the successful functionalization with epoxide. These functionalized SWNTs were used to fabricate nanocomposites, resulting in uniform dispersion and strong interfacial bonding. The mechanical test demonstrated that, with only 1 wt% loading of functionalized SWNTs, the tensile strength of nanocomposites was improved by 40%, and Young's modulus by 60%.These results suggested that efficient load transfer has been achieved through epoxide-grafting. This investigation provided an efficient way to improve the interfacial bonding of multifunctional high-performance nanocomposites for lightweight structure material applications.

  1. Epoxidation of propylene dimers and isomerization of mixtures obtained

    SciTech Connect

    Dobrev, D.M.; Kurtev, K.S.

    1988-05-10

    Mixtures of hexenes are obtained in the dimerization of propylene on a Ziegler catalyst. By the epoxidation of this mixture by organic peroxides, followed by isomerization of the oxides, C/sub 6/ ketones, which are used as solvents, can be obtained. The hexenes were obtained by dimerization of propylene in the presence of a Ni(C/sub 5/H/sub 7/O/sub 2/)/sub 2/-P(C/sub 6/H/sub 5/)/sub 3/-(C/sub 3/H/sub 5/)/sub 2/AlCl catalytic system. The epoxidation was carried with technical grade isopropylbenzyl hydroperoxide (IPBHP). MoO/sub 2/(C/sub 5/H/sub 7/O/sub 2/)/sub 2/ was used as the catalyst. The relative rates of epoxidation of different isomers contained in the dimeric fraction, with respect to 2-methyl-1-pentene, was determined by means of competing reactions.

  2. Remodeling Natural Products: Chemistry and Serine Hydrolase Activity of a Rocaglate-Derived β-Lactone

    PubMed Central

    2015-01-01

    Flavaglines are a class of natural products with potent insecticidal and anticancer activities. β-Lactones are a privileged structural motif found in both therapeutic agents and chemical probes. Herein, we report the synthesis, unexpected light-driven di-epimerization, and activity-based protein profiling of a novel rocaglate-derived β-lactone. In addition to in vitro inhibition of the serine hydrolases ABHD10 and ACOT1/2, the most potent β-lactone enantiomer was also found to inhibit these enzymes, as well as the serine peptidases CTSA and SCPEP1, in PC3 cells. PMID:24447064

  3. Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

    PubMed

    Tsujita, Natsumi; Kuwahara, Hiroyuki; Koyama, Hiroki; Yanaka, Noriyuki; Arakawa, Kenji; Kuniyoshi, Hisato

    2017-05-01

    The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

  4. Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

    DTIC Science & Technology

    1985-07-01

    Stereoselective Reaction of Organophosphinates with Proteinases ........................................... 27 6a. Methods...phenyl)- phosphinate (II) with 0.34 mM proteinases in 0.1 M MOPS buffer, pH 7.5, at 37°C, as monitored by D-DBPG chiral-phase HPLC of extracts. (A...isopropyl(phenyl)phosihinate (III) with 0.34 mM proteinases in 0.1 M MOPS buffer, pH 7.5, at 37*C as monitored by L-DBPG chiral-phase HPLC of extracts

  5. Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates.

    DTIC Science & Technology

    1983-07-01

    filtrate was centrifuged for 10 min at 677 x gravity in a Sorvall RC-5 centrifuge. The reddish-orange supernatant was retained as serum. To precipitate...paraoxon. 21 Paraoxonase fractions were pooled and applied to a column of dye-ligand Matrex Gel Red A0 (Amicon Corporation, Danvers, Massachusetts...9). After Matrex Gel Red A chromatography, paraoxonase excited at 285 nm emitted a peak of fluorescence at 350 nm (Fig. 9) and it absorbed

  6. Synthetic Utility of Epoxides for Chiral Functionalization of Isoxazoles.

    PubMed

    Nelson, Jared K; Burns, Christopher T; Smith, Miles P; Twamley, Brendan; Natale, N R

    2008-05-05

    The lithio-anion of isoxazole 2 was found to ring open propylene oxide in good yields with complete regioselectivity. Vinylic and benzylic epoxides were utilized as key examples of electrophiles and found to produce a mixture of regioisomeric adducts. Additionally, the use of chiral epoxides was explored, and absolute configuration was determined by X-ray crystallography to prove that nucleophilic attack at the benzylic carbon of (R)-styrene oxide proceeds with 100% inversion at the benzylic carbon to afford the (S)-alcohol (4b).

  7. Synthetic Utility of Epoxides for Chiral Functionalization of Isoxazoles

    PubMed Central

    Nelson, Jared K.; Burns, Christopher T.; Smith, Miles P.; Twamley, Brendan; Natale, N. R.

    2010-01-01

    The lithio-anion of isoxazole 2 was found to ring open propylene oxide in good yields with complete regioselectivity. Vinylic and benzylic epoxides were utilized as key examples of electrophiles and found to produce a mixture of regioisomeric adducts. Additionally, the use of chiral epoxides was explored, and absolute configuration was determined by X-ray crystallography to prove that nucleophilic attack at the benzylic carbon of (R)-styrene oxide proceeds with 100% inversion at the benzylic carbon to afford the (S)-alcohol (4b). PMID:21103024

  8. Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification

    SciTech Connect

    Nomura, Daniel K.; Fujioka, Kazutoshi; Issa, Roger S.; Ward, Anna M.; Cravatt, Benjamin F.; Casida, John E.

    2008-04-01

    Serine hydrolase KIAA1363 is an acetyl monoalkylglycerol ether (AcMAGE) hydrolase involved in tumor cell invasiveness. It is also an organophosphate (OP) insecticide-detoxifying enzyme. The key to understanding these dual properties was the use of KIAA1363 +/+ (wildtype) and -/- (gene deficient) mice to define the role of this enzyme in brain and other tissues and its effectiveness in vivo in reducing OP toxicity. KIAA1363 was the primary AcMAGE hydrolase in brain, lung, heart and kidney and was highly sensitive to inactivation by chlorpyrifos oxon (CPO) (IC{sub 50} 2 nM) [the bioactivated metabolite of the major insecticide chlorpyrifos (CPF)]. Although there was no difference in hydrolysis product monoalkylglycerol ether (MAGE) levels in +/+ and -/- mouse brains in vivo, isopropyl dodecylfluorophosphonate (30 mg/kg) and CPF (100 mg/kg) resulted in 23-51% decrease in brain MAGE levels consistent with inhibition of AcMAGE hydrolase activity. On incubating +/+ and -/- brain membranes with AcMAGE and cytidine-5'-diphosphocholine, the absence of KIAA1363 activity dramatically increased de novo formation of platelet-activating factor (PAF) and lyso-PAF, signifying that metabolically-stabilized AcMAGE can be converted to this bioactive lipid in brain. On considering detoxification, KIAA1363 -/- mice were significantly more sensitive than +/+ mice to ip-administered CPF (100 mg/kg) and parathion (10 mg/kg) with increased tremoring and mortality that correlated for CPF with greater brain acetylcholinesterase inhibition. Docking AcMAGE and CPO in a KIAA1363 active site model showed similar positioning of their acetyl and trichloropyridinyl moieties, respectively. This study establishes the relevance of KIAA1363 in ether lipid metabolism and OP detoxification.

  9. Ascorbate-independent carotenoid de-epoxidation in intact spinach chloroplasts.

    PubMed

    Sokolove, P M; Marsho, T V

    1976-05-14

    Slow (greater 1 s) light-induced absorbance changes in the 475-5300 nm spectral region were examined in Type A chloroplasts from spinach. The most prominent absorption change occurred at 505 nm. The difference spectrum for this light-induced increase, its absence in osmotically shocked chloroplasts and restoration by ascorbate, and its sensitivity to dithiothreitol indicate that the absorption change is due to carotenoid de-epoxidatiion. The reaction in intact chloroplasts is characterized by its independence of exogenous ascorbate and a rate constant 3- to 8-fold higher than that reported previously for chloroplasts supplemented with ascorbate. The relevance of carotenoid de-epoxidation to other photosynthetic processes was examined by comparing their sensitivities to dithiothreitol. Levels of dithiothreitol that eliminate the 505 nm shift are without effect on saturated rates of CO2 fixation and do not appreciably inhibit fluorescence quenching. We conclude that carotenoid de-epoxidation is not directly involved in the reactions of photosynthesis or in the regulation of excitation allocation between the photosystems.

  10. Multicomponent linchpin couplings. Reaction of dithiane anions with terminal epoxides, epichlorohydrin, and vinyl epoxides: efficient, rapid, and stereocontrolled assembly of advanced fragments for complex molecule synthesis.

    PubMed

    Smith, Amos B; Pitram, Suresh M; Boldi, Armen M; Gaunt, Matthew J; Sfouggatakis, Chris; Moser, William H

    2003-11-26

    The development, application, and advantages of a one-flask multicomponent dithiane linchpin coupling protocol, over the more conventional stepwise addition of dithiane anions to electrophiles leading to the rapid, efficient, and stereocontrolled assembly of highly functionalized intermediates for complex molecule synthesis, are described. Competent electrophiles include terminal epoxides, epichlorohydrin, and vinyl epoxides. High chemoselectivity can be achieved with epichlorohydrin and vinyl epoxides. For vinyl epoxides, the steric nature of the dithiane anion is critical; sterically unencumbered dithiane anions afford S(N)2 adducts, whereas encumbered anions lead primarily to SN2' adducts. Mechanistic studies demonstrate that the SN2' process occurs via syn addition to the vinyl epoxide. Integration of the multicomponent tactic with epichlorohydrin and vinyl epoxides permits the higher-order union of four and five components.

  11. Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation*

    PubMed Central

    Wang, Yun; Kavran, Jennifer M.; Chen, Zan; Karukurichi, Kannan R.; Leahy, Daniel J.; Cole, Philip A.

    2014-01-01

    S-Adenosylhomocysteine hydrolase (SAHH) is an NAD+-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys401 and Lys408 but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys401 and Lys408 and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD+ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns. PMID:25248746

  12. Effect of short-term exposure to dichlorvos on synaptic plasticity of rat hippocampal slices: Involvement of acylpeptide hydrolase and {alpha}{sub 7} nicotinic receptors

    SciTech Connect

    Olmos, Cristina; Sandoval, Rodrigo; Rozas, Carlos; Navarro, Sebastian; Wyneken, Ursula; Zeise, Marc; Morales, Bernardo; Pancetti, Floria

    2009-07-01

    Dichlorvos is the active molecule of the pro-drug metrifonate used to revert the cognitive deficits associated with Alzheimer's disease. A few years ago it was reported that dichlorvos inhibits the enzyme acylpeptide hydrolase at lower doses than those necessary to inhibit acetylcholinesterase to the same extent. Therefore, the aim of our investigation was to test the hypothesis that dichlorvos can enhance synaptic efficacy through a mechanism that involves acylpeptide hydrolase instead of acetylcholinesterase inhibition. We used long-term potentiation induced in rat hippocampal slices as a model of synaptic plasticity. Our results indicate that short-term exposures (20 min) to 50 {mu}M dichlorvos enhance long-term potentiation in about 200% compared to the control condition. This effect is correlated with approximately 60% inhibition of acylpeptide hydrolase activity, whereas acetylcholinesterase activity remains unaffected. Paired-pulse facilitation and inhibition experiments indicate that dichlorvos does not have any presynaptic effect in the CA3 {yields} CA1 pathway nor affect gabaergic interneurons. Interestingly, the application of 100 nM methyllicaconitine, an {alpha}{sub 7} nicotinic receptor antagonist, blocked the enhancing effect of dichlorvos on long-term potentiation. These results indicate that under the exposure conditions described above, dichlorvos enhances long-term potentiation through a postsynaptic mechanism that involves (a) the inhibition of the enzyme acylpeptide hydrolase and (b) the modulation of {alpha}{sub 7} nicotinic receptors.

  13. Genetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica for removal of methyl parathion in water.

    PubMed

    Wang, Xing-Xing; Chi, Zhe; Ru, Shao-Guo; Chi, Zhen-Ming

    2012-09-01

    In this study, the mph gene encoding methyl parathion hydrolase from Pseudomonas sp. WBC-3 was expressed in Yarrowia lipolytica and the expressed methyl parathion hydrolase was displayed on cell surface of Y. lipolytica. The activity of methyl parathion hydrolase displayed on the yeast cells of the transformant Z51 was 59.5 U mg⁻¹ of cell dry cells (450.6 U per mL of the culture) in the presence of 5.0 mM of Co²⁺. The displayed methyl parathion hydrolase had the optimal pH of 9.5 and the optimal temperature of 40 °C, respectively and was stable in the pH range of 4.5-11 and up to 40 °C. The displayed methyl parathion hydrolase was also stimulated by Co²⁺, Cu²⁺, Ni²⁺ and Mn²⁺, and was not affected by Fe²⁺, Fe³⁺, Na⁺, K⁺, Ca²⁺ and Zn²⁺, but was inhibited by other cations tested. Under the optimal conditions (OD(600 nm) = 2.6, the substrate concentration = 100 mg L⁻¹ and 40 °C), 90.8 % of methyl parathion was hydrolyzed within 30 min. Under the similar conditions, 98.7, 97.0, 96.5 and 94.4 % of methyl parathion in tap water (pH 9.5), tap water (pH 6.8), seawater (pH 9.5) and natural seawater (pH 8.2) were hydrolyzed, respectively, suggesting that the methyl parathion hydrolase displayed on the yeast cells can effectively remove methyl parathion in water.

  14. Visualizing the Mechanism of Epoxide Hydrolysis by the Bacterial Virulence Enzyme Cif.

    PubMed

    Bahl, Christopher D; Hvorecny, Kelli L; Morisseau, Christophe; Gerber, Scott A; Madden, Dean R

    2016-02-09

    The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

  15. Selective N-hydroxyhydantoin carbamate inhibitors of mammalian serine hydrolases

    PubMed Central

    Cognetta, Armand B.; Niphakis, Micah J.; Lee, Hyeon-Cheol; Martini, Michael L.; Hulce, Jonathan J.; Cravatt, Benjamin F.

    2015-01-01

    Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis and screening of a small library of NHH carbamates by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterease-1 (PPT1), mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis. PMID:26120000

  16. Diastereoselective Synthesis of Diketopiperazine Bis-α,β-Epoxides

    PubMed Central

    Ando, Shin; Grote, Amy L.; Koide, Kazunori

    2011-01-01

    Functionalized diketopiperazines (a.k.a. dioxopiperazines) are an important class of molecules in medicinal chemistry and material science. Herein we report a diastereoselective synthesis of diketopiperazine bis-α,β-epoxides via the oxidation of exocyclic olefins. Although six diastereomers may be formed by this approach, only one or two of them were observed. PMID:21250704

  17. ULTRASOUND-ASSISTED ORGANIC SYNTHESIS: ALCOHOL OXIDATION AND OLEFIN EPOXIDATION

    EPA Science Inventory

    Ultrasound-assisted Organic Synthesis: Alcohol Oxidation and Olefin Epoxidation

    Unnikrishnan R Pillai, Endalkachew Sahle-Demessie , Vasudevan Namboodiri, Quiming Zhao, Juluis Enriquez
    U.S. EPA , 26 W. Martin Luther King Dr. , Cincinnati, OH 45268
    Phone: 513-569-773...

  18. Hydrolytic and aminolytic kinetic resolution of terminal bis-epoxides.

    PubMed

    Bredihhina, Jevgenia; Villo, Piret; Andersons, Kārlis; Toom, Lauri; Vares, Lauri

    2013-03-15

    Hydrolytic and aminolytic kinetic resolution of terminal bis-epoxides catalyzed by (salen)Co(III) complexes affords epoxy-diols and N-protected epoxy-amino alcohols with excellent enantio- and diastereoselectivity and good yields. An operationally simple procedure gives instant access to valuable building blocks containing two remote stereocenters in highly enantioenriched form.

  19. Ring-opening Polymerization of Epoxidized Soybean Oil

    USDA-ARS?s Scientific Manuscript database

    Ring opening polymerization of epoxidized soybean oil (ESO) initiated by boron trifluoride diethyl etherate, (BF3•OEt2), in methylene chloride was conducted in an effort to develop useful biodegradable polymers. The resulting polymers (PESO) were characterized using Infrared (IR), differential scan...

  20. Lutein epoxide cycle, more than just a forest tale

    PubMed Central

    Becerril, José María; García-Plazaola, José Ignacio

    2009-01-01

    Two xanthophyll cycles have been described in higher plants: the ubiquitous violaxanthin (V) cycle and the taxonomically restricted lutein epoxide (Lx) cycle. Both involve the light induced de-epoxidation of an epoxidated xanthophyll (V or Lx) and the epoxidation back in the dark. Evolutionary trends and function of the Lx cycle are still not clear. Up to nowadays, significant amounts of Lx have been found in several unrelated taxa, but it is a character almost exclusive from woody plants (except in the case of the parasitic plant Cuscuta reflexa). We have found an exception to this pattern in Cucumis sativus L., which showed high concentrations of Lx. Since Lx cycle was operative in leaves and cotyledons of this species and Lx concentration were much higher in cotyledons than in leaves, we speculate a role for the early stages of development. To date, this species is the first herbaceous non-parasitic species with operative Lx cycle. Since this species can be much more easily and rapidly grown and investigated than woody plants, these data can open new horizons and new lines of investigation for Lx cycle. PMID:19794858

  1. Epoxidation of aldrin to exo-dieldrin by soil bacteria.

    PubMed Central

    Ferguson, J A; Korte, F

    1977-01-01

    Twenty-two strains of soil bacteria, including representatives of the genera Bacillus, Micromonospora, Mycobacterium, Nocardia, Streptomyces, Thermoactinomyces, and Pseudomonas and 10 unidentified gram-negative, motile, rod-shaped bacteria, were shown to degrade aldrin to its epoxide dieldrin. In every case, the exo-stereoisomer of dieldrin was produced exclusively. PMID:407844

  2. ULTRASOUND-ASSISTED ORGANIC SYNTHESIS: ALCOHOL OXIDATION AND OLEFIN EPOXIDATION

    EPA Science Inventory

    Ultrasound-assisted Organic Synthesis: Alcohol Oxidation and Olefin Epoxidation

    Unnikrishnan R Pillai, Endalkachew Sahle-Demessie , Vasudevan Namboodiri, Quiming Zhao, Juluis Enriquez
    U.S. EPA , 26 W. Martin Luther King Dr. , Cincinnati, OH 45268
    Phone: 513-569-773...

  3. 3,3',4,4',5-Pentachlorobiphenyl (PCB 126) Decreases Hepatic and Systemic Ratios of Epoxide to Diol Metabolites of Unsaturated Fatty Acids in Male Rats.

    PubMed

    Wu, Xianai; Yang, Jun; Morisseau, Christophe; Robertson, Larry W; Hammock, Bruce; Lehmler, Hans-Joachim

    2016-08-01

    Disruption of the homeostasis of oxygenated regulatory lipid mediators (oxylipins), potential markers of exposure to aryl hydrocarbon receptor (AhR) agonists, such as 3,3',4,4',5-pentachlorobiphenyl (PCB 126), is associated with a range of diseases, including nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Here we test the hypothesis that PCB 126 exposure alters the levels of oxylipins in rats. Male Sprague-Dawley rats (5-weeks old) were treated over a 3-month period every 2 weeks with intraperitoneal injections of PCB 126 in corn oil (cumulative doses of 0, 19.8, 97.8, and 390 µg/kg b.w.; 6 injections total). PCB 126 treatment caused a reduction in growth rates at the highest dose investigated, a dose-dependent decrease in thymus weights, and a dose-dependent increase in liver weights. Liver PCB 126 levels increased in a dose-dependent manner, while levels in plasma were below or close to the detection limit. The ratios of several epoxides to diol metabolites formed via the cytochrome P450 (P450) monooxygenase/soluble epoxide hydrolase (sEH) pathway from polyunsaturated fatty acids displayed a dose-dependent decrease in the liver and plasma, whereas levels of oxylipins formed by other metabolic pathways were generally not altered by PCB 126 treatment. The effects of PCB 126 on epoxide-to-diol ratios were associated with an increased CYP1A activity in liver microsomes and an increased sEH activity in liver cytosol and peroxisomes. These results suggest that oxylipins are potential biomarkers of exposure to PCB 126 and that the P450/sEH pathway is a therapeutic target for PCB 126-mediated hepatotoxicity that warrants further attention. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. for Permissions, please e-mail: journals.permissions@oup.com.

  4. Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase.

    PubMed

    Chen, Xiabin; Huang, Xiaoqin; Geng, Liyi; Xue, Liu; Hou, Shurong; Zheng, Xirong; Brimijoin, Stephen; Zheng, Fang; Zhan, Chang-Guo

    2015-03-01

    Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A¹⁹⁹S/S²²⁷A/S²⁸⁷G/A³²⁸W/Y³³²G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A¹⁹⁹S/F²²⁷A/S²⁸⁷G/A³²⁸W/Y³³²G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (k(cat)/K(M)) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.

  5. Bacterial growth does require peptidoglycan hydrolases.

    PubMed

    Vollmer, Waldemar

    2012-12-01

    Most bacteria surround their cytoplasmic membrane with a net-like, elastic heteropolymer, the peptidoglycan sacculus, to protect themselves from bursting due to the turgor and to maintain cell shape. It has been assumed that growing bacteria require peptidoglycan hydrolases to open meshes in the peptidoglycan net allowing the insertion of the newly synthesized material for surface expansion. However, peptidoglycan hydrolases essential for bacterial growth have long remained elusive. In this issue of Molecular Microbiology Singh et al. (2012) report the identification in Escherichia coli of three new DD-endopeptidases (Spr, YdhO and YebA) which are collectively required for peptidoglycan growth. Cells depleted of the three enzymes fail to incorporate new peptidoglycan, indicating that the cleavage of cross-links by the new endopeptidases is needed for surface growth of the sacculus. These results are corroborated by recent data showing that Bacillus subtilis cells require the DL-endopeptidase activity of CwlO or LytE for growth.

  6. Identification of Neutral Cholesterol Ester Hydrolase, a Key Enzyme Removing Cholesterol from Macrophages*S⃞

    PubMed Central

    Okazaki, Hiroaki; Igarashi, Masaki; Nishi, Makiko; Sekiya, Motohiro; Tajima, Makiko; Takase, Satoru; Takanashi, Mikio; Ohta, Keisuke; Tamura, Yoshiaki; Okazaki, Sachiko; Yahagi, Naoya; Ohashi, Ken; Amemiya-Kudo, Michiyo; Nakagawa, Yoshimi; Nagai, Ryozo; Kadowaki, Takashi; Osuga, Jun-ichi; Ishibashi, Shun

    2008-01-01

    Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis. PMID:18782767

  7. Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca.

    PubMed

    Dani, M P; Edwards, J P; Richards, E H

    2005-07-01

    Venom from the pupal endoparasitoid, Pimpla hypochondriaca has previously been shown to contain a mixture of biologically active molecules. Currently, P. hypochondriaca venom was examined for the presence of hydrolase activity. Six hydrolases were consistently detected using the API ZYM semiquantitative colourimetric kit. The main hydrolases detected were; acid phosphatase, beta-glucosidase, esterase, beta-galactosidase, esterase lipase, and lipase. The most rapid and intense colour reaction was detected for acid phosphatase. The pH optimum and the specific activity of venom acid phosphatase was determined using p-nitrophenol phosphate as a substrate and were 4.8 and 0.47 nmol p-nitrophenol/min/microg of venom protein, respectively. The acid phosphatase activity was inhibited in a dose dependent manner by sodium fluoride (IC(50) 4.2 x 10(-4) M), and by cocktail inhibitor 2 (CI 2). P. hypochondriaca venom has previously been shown to display potent cytotoxic activity towards Lacanobia oleracea haemocytes maintained in vitro. The contribution of acid phosphatase in venom to this cytotoxic activity was investigated by titrating venom against CI 2 prior to the addition of L. oleracea haemocytes. The results suggest that, despite the relatively high levels of acid phosphatase activity in venom, venom acid phosphatase plays no role in the antihaemocytic activity of P. hypochondriaca venom in vitro.

  8. Effect of epoxidation level on thermal properties and ionic conductivity of epoxidized natural rubber solid polymer nanocomposite electrolytes

    SciTech Connect

    Harun, Fatin; Chan, Chin Han; Winie, Tan; Sim, Lai Har; Zainal, Nurul Fatahah Asyqin

    2015-08-28

    Effect of epoxide content on the thermal and conductivity properties of epoxidized natural rubber (ENR) solid polymer nanocomposite electrolytes was investigated. Commercial available epoxidized natural rubber having 25 (ENR25) and 50 mole% (ENR50) epoxide, respectively were incorporated with lithium perchlorate (LiClO{sub 4}) salt and titanium dioxide (TiO{sub 2}) nanofiller via solution casting method. The solid polymer nanocomposite electrolytes were characterized by differential scanning calorimetry (DSC) and impedance spectroscopy (IS) for their thermal properties and conductivity, respectively. It was evident that introduction of LiClO{sub 4} causes a greater increase in glass transition temperature (T{sub g}) and ionic conductivity of ENR50 as compared to ENR25. Upon addition of TiO{sub 2} in ENR/LiClO{sub 4} system, a remarkable T{sub g} elevation was observed for both ENRs where ENR50 reveals a more pronounced changes. It is interesting to note that they exhibit different phenomenon in ionic conductivity with TiO{sub 2} loading where ENR25 shows enhancement of conductivity while ENR50 shows declination.

  9. Effect of epoxidation level on thermal properties and ionic conductivity of epoxidized natural rubber solid polymer nanocomposite electrolytes

    NASA Astrophysics Data System (ADS)

    Harun, Fatin; Chan, Chin Han; Sim, Lai Har; Winie, Tan; Zainal, Nurul Fatahah Asyqin

    2015-08-01

    Effect of epoxide content on the thermal and conductivity properties of epoxidized natural rubber (ENR) solid polymer nanocomposite electrolytes was investigated. Commercial available epoxidized natural rubber having 25 (ENR25) and 50 mole% (ENR50) epoxide, respectively were incorporated with lithium perchlorate (LiClO4) salt and titanium dioxide (TiO2) nanofiller via solution casting method. The solid polymer nanocomposite electrolytes were characterized by differential scanning calorimetry (DSC) and impedance spectroscopy (IS) for their thermal properties and conductivity, respectively. It was evident that introduction of LiClO4 causes a greater increase in glass transition temperature (Tg) and ionic conductivity of ENR50 as compared to ENR25. Upon addition of TiO2 in ENR/LiClO4 system, a remarkable Tg elevation was observed for both ENRs where ENR50 reveals a more pronounced changes. It is interesting to note that they exhibit different phenomenon in ionic conductivity with TiO2 loading where ENR25 shows enhancement of conductivity while ENR50 shows declination.

  10. Bacterial CS2 Hydrolases from Acidithiobacillus thiooxidans Strains Are Homologous to the Archaeal Catenane CS2 Hydrolase

    PubMed Central

    Smeulders, Marjan J.; Pol, Arjan; Venselaar, Hanka; Barends, Thomas R. M.; Hermans, John; Jetten, Mike S. M.

    2013-01-01

    Carbon disulfide (CS2) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS2 is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO2) and hydrogen sulfide (H2S), a property used in industrial biofiltration of CS2-polluted airstreams. We report on the mechanism of bacterial CS2 conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS2 hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS2 hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of β-carbonic anhydrase (β-CA) homologues that may comprise a subclass of CS2 hydrolases within the β-CA family. Unlike CAs, the CS2 hydrolases did not hydrate CO2 but converted CS2 and COS with H2O to H2S and CO2. The CS2 hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS2 hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS2 hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS2 hydrolases based on the structure of Acidianus strain A1-3 CS2 hydrolase suggest that the A. thiooxidans strain G8 CS2 hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation. PMID:23836868

  11. The fatty-acid amide hydrolase inhibitor URB597 does not affect triacylglycerol hydrolysis in rat tissues.

    PubMed

    Clapper, Jason R; Duranti, Andrea; Tontini, Andrea; Mor, Marco; Tarzia, Giorgio; Piomelli, Daniele

    2006-11-01

    The O-arylcarbamate URB597 (cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester; also referred to as KDS-4103) is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the inactivation of the endogenous cannabinoid anandamide. URB597 demonstrates a remarkable degree of selectivity for FAAH over other serine hydrolases (e.g. cholinesterases) or other components of the endocannabinoid system (e.g. cannabinoid receptors). However, in a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain proteins, it was recently shown that URB597 prevents FPR binding to triacylglycerol hydrolase (TGH) with a median inhibitory concentration of 192nM. To determine whether this effect correlates with inhibition of TGH activity, we investigated the ability of URB597 to inhibit triolein hydrolysis in rat liver and heart tissues, which are rich in TGH, as well as white adipose tissue (WAT), which is rich in adipose triacylglycerol lipase (TGL) and hormone-sensitive lipase. The results show that URB597 does not affect triolein hydrolysis in any of these tissues at concentrations as high as 10microM, whereas it inhibits FAAH activity at low nanomolar concentrations. Moreover, intraperitoneal (i.p.) administration of URB597 at doses that maximally inhibit FAAH in vivo (0.3-3mgkg(-1)) exerts no effect on triolein hydrolysis and tissue triacylglycerol (TAG) levels in rat liver, heart or WAT. The results indicate that URB597, while potent at inhibiting FAAH, does not affect TGH and TGL activities in rat tissues.

  12. A computational study of acid catalyzed aerosol reactions of atmospherically relevant epoxides.

    PubMed

    Piletic, Ivan R; Edney, Edward O; Bartolotti, Libero J

    2013-11-07

    Epoxides are important intermediates of atmospheric isoprene oxidation. Their subsequent reactions in the particle phase lead to the production of organic compounds detected in ambient aerosols. We apply density functional theory to determine the important kinetic factors that drive epoxide reactions in the particle phase. Specifically, the importance of acid catalysis and solvent polarity are investigated using a variety of epoxides and nucleophiles. The condensed phase is modeled using molecular clusters immersed in a dielectric continuum and a majority of the calculations are performed with the M062x density functional and the 6-311++G** basis set. Calculations of acid catalyzed epoxide hydrolysis transition states for simple primary, secondary and tertiary epoxides are consistent with an A-2 mechanism where the nucleophile (water) interacts with an epoxide carbon in the transition state. By applying transition state theory to this mechanism, the overall rate constants of epoxide reactions such as hydrolysis, organosulfate formation, organonitrate formation and oligomerization are determined. The calculations indicate that the acid catalyzed hydrolysis rate constant of 2-methyl-2,3-epoxybutane-1,4-diol (β-IEPOX--an isoprene epoxide produced under low NOx conditions) is approximately 30 times greater than 2-methyl-2,3-epoxypropanoic acid (MAE--methacrylic acid epoxide derived from isoprene and produced at high NOx concentrations). Furthermore, acid catalyzed organosulfate formation and epoxide oligomerization reactions are competitive and appear to be kinetically favorable over the hydrolysis of IEPOX.

  13. Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System ▿

    PubMed Central

    Yang, Chao; Freudl, Roland; Qiao, Chuanling; Mulchandani, Ashok

    2010-01-01

    A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell. PMID:19933341

  14. Modeling Epoxidation of Drug-like Molecules with a Deep Machine Learning Network

    PubMed Central

    2015-01-01

    Drug toxicity is frequently caused by electrophilic reactive metabolites that covalently bind to proteins. Epoxides comprise a large class of three-membered cyclic ethers. These molecules are electrophilic and typically highly reactive due to ring tension and polarized carbon–oxygen bonds. Epoxides are metabolites often formed by cytochromes P450 acting on aromatic or double bonds. The specific location on a molecule that undergoes epoxidation is its site of epoxidation (SOE). Identifying a molecule’s SOE can aid in interpreting adverse events related to reactive metabolites and direct modification to prevent epoxidation for safer drugs. This study utilized a database of 702 epoxidation reactions to build a model that accurately predicted sites of epoxidation. The foundation for this model was an algorithm originally designed to model sites of cytochromes P450 metabolism (called XenoSite) that was recently applied to model the intrinsic reactivity of diverse molecules with glutathione. This modeling algorithm systematically and quantitatively summarizes the knowledge from hundreds of epoxidation reactions with a deep convolution network. This network makes predictions at both an atom and molecule level. The final epoxidation model constructed with this approach identified SOEs with 94.9% area under the curve (AUC) performance and separated epoxidized and non-epoxidized molecules with 79.3% AUC. Moreover, within epoxidized molecules, the model separated aromatic or double bond SOEs from all other aromatic or double bonds with AUCs of 92.5% and 95.1%, respectively. Finally, the model separated SOEs from sites of sp2 hydroxylation with 83.2% AUC. Our model is the first of its kind and may be useful for the development of safer drugs. The epoxidation model is available at http://swami.wustl.edu/xenosite. PMID:27162970

  15. Modeling Epoxidation of Drug-like Molecules with a Deep Machine Learning Network.

    PubMed

    Hughes, Tyler B; Miller, Grover P; Swamidass, S Joshua

    2015-07-22

    Drug toxicity is frequently caused by electrophilic reactive metabolites that covalently bind to proteins. Epoxides comprise a large class of three-membered cyclic ethers. These molecules are electrophilic and typically highly reactive due to ring tension and polarized carbon-oxygen bonds. Epoxides are metabolites often formed by cytochromes P450 acting on aromatic or double bonds. The specific location on a molecule that undergoes epoxidation is its site of epoxidation (SOE). Identifying a molecule's SOE can aid in interpreting adverse events related to reactive metabolites and direct modification to prevent epoxidation for safer drugs. This study utilized a database of 702 epoxidation reactions to build a model that accurately predicted sites of epoxidation. The foundation for this model was an algorithm originally designed to model sites of cytochromes P450 metabolism (called XenoSite) that was recently applied to model the intrinsic reactivity of diverse molecules with glutathione. This modeling algorithm systematically and quantitatively summarizes the knowledge from hundreds of epoxidation reactions with a deep convolution network. This network makes predictions at both an atom and molecule level. The final epoxidation model constructed with this approach identified SOEs with 94.9% area under the curve (AUC) performance and separated epoxidized and non-epoxidized molecules with 79.3% AUC. Moreover, within epoxidized molecules, the model separated aromatic or double bond SOEs from all other aromatic or double bonds with AUCs of 92.5% and 95.1%, respectively. Finally, the model separated SOEs from sites of sp(2) hydroxylation with 83.2% AUC. Our model is the first of its kind and may be useful for the development of safer drugs. The epoxidation model is available at http://swami.wustl.edu/xenosite.

  16. Recognition of Corn Defense Chitinases by Fungal Polyglycine Hydrolases.

    PubMed

    Naumann, Todd A; Bakota, Erica L; Price, Neil P J

    2017-04-06

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly-Gly bonds within the polyglycine region. Some Gly-Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly-Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es-cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly->sarcosine (N-methylglycine) mutations or a specific Ser->Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses. This article is protected by copyright. All rights reserved.

  17. Glycoside Hydrolases across Environmental Microbial Communities

    PubMed Central

    Berlemont, Renaud

    2016-01-01

    Across many environments microbial glycoside hydrolases support the enzymatic processing of carbohydrates, a critical function in many ecosystems. Little is known about how the microbial composition of a community and the potential for carbohydrate processing relate to each other. Here, using 1,934 metagenomic datasets, we linked changes in community composition to variation of potential for carbohydrate processing across environments. We were able to show that each ecosystem-type displays a specific potential for carbohydrate utilization. Most of this potential was associated with just 77 bacterial genera. The GH content in bacterial genera is best described by their taxonomic affiliation. Across metagenomes, fluctuations of the microbial community structure and GH potential for carbohydrate utilization were correlated. Our analysis reveals that both deterministic and stochastic processes contribute to the assembly of complex microbial communities. PMID:27992426

  18. Localization of Acid Hydrolases in Protoplasts

    PubMed Central

    Butcher, Henry C.; Wagner, George J.; Siegelman, Harold W.

    1977-01-01

    The development of techniques to isolate and purify relatively large quantities of intact vacuoles from mature tissues permits direct biochemical analysis of this ubiquitous mature plant cell organelle. Vacuoles and a fraction enriched in soluble cytoplasmic constituents were quantitatively prepared from Hippeastrum flower petal protoplasts. Vacuolar lysate and soluble cytoplasmic fractions were examined for acid hydrolase activities commonly associated with animal lysosomes, and pH optima were determined. Esterase, protease, carboxypeptidase, β-galactosidase, α-glycosidase and β-glycosidase, not found in the vacuole lysate fraction, were components of the soluble cytoplasmic fraction. Acid phosphatase, RNase and DNase were present in both fractions. Vacuolar enzyme activities were also examined as a function of flower development from bud through senescent stages. The data obtained are not consistent with the concept that the mature plant cell vacuole functions as a generalized lysosome. PMID:16660001

  19. A simplified electrostatic model for hydrolase catalysis.

    PubMed

    Pessoa Filho, Pedro de Alcantara; Prausnitz, John M

    2015-07-01

    Toward the development of an electrostatic model for enzyme catalysis, the active site of the enzyme is represented by a cavity whose surface (and beyond) is populated by electric charges as determined by pH and the enzyme's structure. The electric field in the cavity is obtained from electrostatics and a suitable computer program. The key chemical bond in the substrate, at its ends, has partial charges with opposite signs determined from published force-field parameters. The electric field attracts one end of the bond and repels the other, causing bond tension. If that tension exceeds the attractive force between the atoms, the bond breaks; the enzyme is then a successful catalyst. To illustrate this very simple model, based on numerous assumptions, some results are presented for three hydrolases: hen-egg white lysozyme, bovine trypsin and bovine ribonuclease. Attention is given to the effect of pH.

  20. Structure of Francisella tularensis peptidyl-tRNA hydrolase

    PubMed Central

    Clarke, Teresa E.; Romanov, Vladimir; Lam, Robert; Gothe, Scott A.; Peddi, Srinivasa R.; Razumova, Ekaterina B.; Lipman, Richard S. A.; Branstrom, Arthur A.; Chirgadze, Nickolay Y.

    2011-01-01

    The rational design of novel antibiotics for bacteria involves the identification of inhibitors for enzymes involved in essential biochemical pathways in cells. In this study, the cloning, expression, purification, crystallization and structure of the enzyme peptidyl-tRNA hydrolase from Francisella tularensis, the causative agent of tularemia, was performed. The structure of F. tularensis peptidyl-tRNA hydrolase is comparable to those of other bacterial peptidyl-tRNA hydrolases, with most residues in the active site conserved amongst the family. The resultant reagents, structural data and analyses provide essential information for the structure-based design of novel inhibitors for this class of proteins. PMID:21505237

  1. A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films.

    PubMed

    Barth, Markus; Honak, Annett; Oeser, Thorsten; Wei, Ren; Belisário-Ferrari, Matheus R; Then, Johannes; Schmidt, Juliane; Zimmermann, Wolfgang

    2016-08-01

    TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.

  2. Differential inhibition of aflatoxin B1 oxidation by gestodene action on human liver microsomes.

    PubMed

    Kim, B R; Oh, H S; Kim, D H

    1997-11-01

    Human cytochrome P450 (P450) 3A is known to be involved in the formation of both aflatoxin B1-exo-8,9-epoxide (exo-epoxidation) and aflatoxin Q1 (3 alpha-hydroxylation). Gestodene, a known inactivator of P450 3A4, inhibited the formation of AFB1 metabolites in a variety of ways depending on the incubation condition. Preincubation of gestodene with human liver microsomes prior to the addition of AFB1 inhibited both exo-epoxidation and 3 alpha-hydroxylation whereas simultaneous incubation of gestodene with AFB1 only inhibited 3 alpha-hydroxylation. These results suggest that two independent substrate binding sites exist in P450 3A4, and AFB1 binds to both of the binding sites. Gestodene selectively binds to one of the binding sites leading to the formation of AFQ1, whereas it does not affect the formation of exo-epoxide via the other binding site.

  3. Discovery of MK-3168: A PET Tracer for Imaging Brain Fatty Acid Amide Hydrolase

    PubMed Central

    2013-01-01

    We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [11C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain. PMID:24900701

  4. Aryl Piperazinyl Ureas as Inhibitors of Fatty Acid Amide Hydrolase (FAAH) in Rat, Dog, and Primate.

    PubMed

    Keith, John M; Apodaca, Rich; Tichenor, Mark; Xiao, Wei; Jones, William; Pierce, Joan; Seierstad, Mark; Palmer, James; Webb, Michael; Karbarz, Mark; Scott, Brian; Wilson, Sandy; Luo, Lin; Wennerholm, Michelle; Chang, Leon; Brown, Sean; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra; Breitenbucher, J Guy

    2012-10-11

    A series of aryl piperazinyl ureas that act as covalent inhibitors of fatty acid amide hydrolase (FAAH) is described. A potent and selective (does not inhibit FAAH-2) member of this class, JNJ-40355003, was found to elevate the plasma levels of three fatty acid amides: anandamide, oleoyl ethanolamide, and palmitoyl ethanolamide, in the rat, dog, and cynomolgous monkey. The elevation of the levels of these lipids in the plasma of monkeys suggests that FAAH-2 may not play a significant role in regulating plasma levels of fatty acid ethanolamides in primates.

  5. Synthesis and biochemical evaluation of guanidino-alkyl-ribitol derivatives as nucleoside hydrolase inhibitors.

    PubMed

    Goeminne, A; McNaughton, M; Bal, G; Surpateanu, G; Van Der Veken, P; De Prol, S; Versées, W; Steyaert, J; Haemers, A; Augustyns, K

    2008-02-01

    Nucleoside hydrolase (NH) is a key enzyme in the purine salvage pathway. The purine specificity of the IAG-NH from Trypanosoma vivax is at least in part due to cation-pi-stacking interactions. Guanidinium ions can be involved in cation-pi-stacking interactions, therefore a series of guanidino-alkyl-ribitol derivatives were synthesized in order to examine the binding affinity of these compounds towards the target enzyme. The compounds show moderate to good inhibiting activity towards the IAG-NH from T. vivax.

  6. Discovery of MK-3168: A PET Tracer for Imaging Brain Fatty Acid Amide Hydrolase.

    PubMed

    Liu, Ping; Hamill, Terence G; Chioda, Marc; Chobanian, Harry; Fung, Selena; Guo, Yan; Chang, Linda; Bakshi, Raman; Hong, Qingmei; Dellureficio, James; Lin, Linus S; Abbadie, Catherine; Alexander, Jessica; Jin, Hong; Mandala, Suzanne; Shiao, Lin-Lin; Li, Wenping; Sanabria, Sandra; Williams, David; Zeng, Zhizhen; Hajdu, Richard; Jochnowitz, Nina; Rosenbach, Mark; Karanam, Bindhu; Madeira, Maria; Salituro, Gino; Powell, Joyce; Xu, Ling; Terebetski, Jenna L; Leone, Joseph F; Miller, Patricia; Cook, Jacquelynn; Holahan, Marie; Joshi, Aniket; O'Malley, Stacey; Purcell, Mona; Posavec, Diane; Chen, Tsing-Bau; Riffel, Kerry; Williams, Mangay; Hargreaves, Richard; Sullivan, Kathleen A; Nargund, Ravi P; DeVita, Robert J

    2013-06-13

    We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.

  7. Pyrazole phenylcyclohexylcarbamates as inhibitors of human fatty acid amide hydrolases (FAAH).

    PubMed

    Aghazadeh Tabrizi, Mojgan; Baraldi, Pier Giovanni; Ruggiero, Emanuela; Saponaro, Giulia; Baraldi, Stefania; Romagnoli, Romeo; Martinelli, Adriano; Tuccinardi, Tiziano

    2015-06-05

    Fatty acid amide hydrolase (FAAH) inhibitors have gained attention as potential therapeutic targets in the management of neuropathic pain. Here, we report a series of pyrazole phenylcyclohexylcarbamate derivatives standing on the known carbamoyl FAAH inhibitor URB597. Structural modifications led to the recognition of compound 22 that inhibited human recombinant FAAH (hrFAAH) in the low nanomolar range (IC50 = 11 nM). The most active compounds of this series showed significant selectivity toward monoacylglycerol lipase (MAGL) enzyme. In addition, molecular modeling and reversibility behavior of the new class of FAAH inhibitors are presented in this article.

  8. Characterization of 2-Bromoethanesulfonate as a Selective Inhibitor of the Coenzyme M-Dependent Pathway and Enzymes of Bacterial Aliphatic Epoxide Metabolism▿

    PubMed Central

    Boyd, Jeffrey M.; Ellsworth, Ashley; Ensign, Scott A.

    2006-01-01

    Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO2 fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO2-fixing enzyme 2-KPCC. PMID:16997966

  9. Asymmetric catalysis with water: efficient kinetic resolution of terminal epoxides by means of catalytic hydrolysis.

    PubMed

    Tokunaga, M; Larrow, J F; Kakiuchi, F; Jacobsen, E N

    1997-08-15

    Epoxides are versatile building blocks for organic synthesis. However, terminal epoxides are arguably the most important subclass of these compounds, and no general and practical method exists for their production in enantiomerically pure form. Terminal epoxides are available very inexpensively as racemic mixtures, and kinetic resolution is an attractive strategy for the production of optically active epoxides, given an economical and operationally simple method. Readily accessible synthetic catalysts (chiral cobalt-based salen complexes) have been used for the efficient asymmetric hydrolysis of terminal epoxides. This process uses water as the only reagent, no added solvent, and low loadings of a recyclable catalyst (<0.5 mole percent), and it affords highly valuable terminal epoxides and 1, 2-diols in high yield with high enantiomeric enrichment.

  10. Styrene-butadiene rubber/halloysite nanotubes composites modified by epoxidized natural rubber.

    PubMed

    Jia, Zhixin; Luo, Yuanfang; Yang, Shuyan; Du, Mingliang; Guo, Baochun; Jia, Demin

    2011-12-01

    The reinforcement effects of halloysite nanotubes on styrene-butadiene rubber and the modification effect of epoxidized natural rubber on styrene-butadiene rubber/halloysite nanotubes composites were studied. The structure, morphology and properties of styrene-butadiene rubber/halloysite nanotubes composites before and after the incorporation of epoxidized natural rubber were investigated. The results indicated that epoxidized natural rubber can promote the dispersion and orientation of halloysite nanotubes in styrene-butadiene rubber matrix at nanoscale and strengthen interfacial combination between halloysite nanotubes and styrene-butadiene rubber by the formation of covalent bonds and hydrogen bonds between epoxidized natural rubber and halloysite nanotubes. Consequently epoxidized natural rubber can improve the mechanical properties of the vulcanizates of styrene-butadiene rubber/halloysite nanotubes composites. Besides epoxidized natural rubber can decrease the rolling resistance of the vulcanizates and increase the wet grip property of the vulcanizates.

  11. Epoxidized natural rubber: Exploring the potential of an old elastomer

    NASA Astrophysics Data System (ADS)

    Mascia, Leno; Russo, Pietro; Lavorgna, Marino; Verdolotti, Letizia; Clarke, Jane; Vignali, Adriano; Acierno, Domenico

    2014-05-01

    A study was carried out to evaluate the efficiency of dodecyl succinic anhydride as a curing agent for a commercial grade of natural rubber that had been epoxidized to approximately 50 %mol (ENR50). It was shown that the maximum achievable gel content for this system is about 87 - 88 %wt due to the presence of non-functionalized species. The incorporation of unmodified natural rubber in the mix reduced the gel content in direct correlation with the dilution of the epoxidized component. Mixing the system, even under "mild" thermal conditions induces rapid gelation due to the high functionality of ENR50. The catalytic effect of N,N-Dimethylbenzylamine was confirmed by both thermal analysis and the curometer evaluations. A quantitative analysis of the latter data has shown that mixing under severe conditions can lead to an increase in reactivity in the subsequent curing step.

  12. Direct Epoxidation of Propylene over Stabilized Cu+ Surface Sites on Ti Modified Cu2O

    DOE PAGES

    Yang, X.; Kattel, S.; Xiong, K.; ...

    2015-07-17

    Direct propylene epoxidation by O2 is a challenging reaction because of the strong tendency for complete combustion. Results from the current study demonstrate the feasibility to tune the epoxidation selectivity by generating highly dispersed and stabilized Cu+ active sites in a TiCuOx mixed oxide. The TiCuOx surface anchors the key surface intermediate, oxametallacycle, leading to higher selectivity for epoxidation of propylene.

  13. Acetylleucine chloromethyl ketone, an inhibitor of acylpeptide hydrolase, induces apoptosis of U937 cells.

    PubMed

    Yamaguchi, M; Kambayashi, D; Toda, J; Sano, T; Toyoshima, S; Hojo, H

    1999-09-16

    Acetylleucine chloromethyl ketone (ALCK), an inhibitor of acylpeptide hydrolase (ACPH), inhibited the growth of human monoblastic U937 cells in a dose- and time-dependent manner. Morphology of the ALCK-exposed cells showed typical apoptosis, judging from the nuclear condensation and segmentation. Chromosomal DNA of U937 cells treated with ALCK showed an internucleosomal ladder-like pattern on electrophoresis, being characteristic of apoptosis. Of the other leucine chloromethyl ketone analogues, butyloylleucine chloromethyl ketone (BLCK) induced a weak ladder-like formation but caploylleucine chloromethyl ketone (CLCK)barely did. On the other hand, intracellular ACPH activity of U937 cells was strongly inhibited by culturing with ALCK, moderately with BLCK, and not with CLCK. These findings indicate that the inhibition of ACPH activity leads to apoptosis and suggest that ACPH may play a vital role in eukaryotic cells. Copyright 1999 Academic Press.

  14. Coordination of oxidized protein hydrolase and the proteasome in the clearance of cytotoxic denatured proteins.

    PubMed

    Shimizu, Kei; Kiuchi, Yukari; Ando, Ken; Hayakawa, Makio; Kikugawa, Kiyomi

    2004-11-05

    Intracellular accumulation of denatured proteins impairs cellular function. The proteasome is recognized as an enzyme responsible for the effective clearance of those cytotoxic denatured proteins. As another enzyme that participates in the destruction of damaged proteins, we have identified oxidized protein hydrolase (OPH) and found that OPH confers cellular resistance to various kinds of oxidative stress. In this study, we demonstrate the roles of the proteasome and OPH in the clearance of denatured proteins. The inhibition of proteasome activity results in the elevation of protein carbonyls in cells under oxidative stress. On the other hand, cells overexpressing OPH retain higher resistance to oxidative stress, even though the proteasome activity is inhibited. Furthermore, upon inhibition of the proteasome activity, OPH is recruited to a novel organelle termed the aggresome where misfolded or denatured proteins are processed. Thus, OPH and the proteasome coordinately contribute to the clearance of cytotoxic denatured proteins.

  15. Inhibition of aryl hydrocarbon receptor transactivation and DNA adduct formation by CYP1 isoform-selective metabolic deactivation of benzo[a]pyrene

    SciTech Connect

    Endo, Kaori; Uno, Shigeyuki; Seki, Taiichiro; Ariga, Toyohiko; Kusumi, Yoshiaki; Mitsumata, Masako; Yamada, Sachiko; Makishima, Makoto

    2008-07-15

    Benzo[a]pyrene (BaP), a polyaromatic hydrocarbon produced by the combustion of cigarettes and coke ovens, is a known procarcinogen. BaP activates the aryl hydrocarbon receptor (AhR) and induces the expression of a battery of genes, including CYP1A1, which metabolize BaP to toxic compounds. The possible role of CYP1 enzymes in mediating BaP detoxification or metabolic activation remains to be elucidated. In this study, we assessed the effects of CYP1 enzymes (CYP1A1, CYP1A2 and CYP1B1) on BaP-induced AhR transactivation and DNA adduct formation in HEK293 cells and HepG2 cells. Transfection of CYP1A1 and CYP1B1, but not CYP1A2, suppressed BaP-induced activation of AhR. Expression of CYP1A1 and CYP1A2, but not CYP1B1, inhibited DNA adduct formation in BaP-treated HepG2 cells. These results indicate that CYP1A1 and CYP1B1 play a role in deactivation of BaP on AhR and that CYP1A1 and CYP1A2 are involved in BaP detoxification by suppressing DNA adduct formation. BaP treatment did not induce DNA adduct formation in HEK293 cells, even after transfection of CYP1 enzymes, suggesting that expression of CYP1 enzymes is not sufficient for DNA adduct formation. Lower expression of epoxide hydrolase and higher expression of glutathione S-transferase P1 (GSTP1) and GSTM1/M2 were observed in HEK293 cells compared with HepG2 cells. Dynamic expression of CYP1A1, CYP1A2 and CYP1B1 along with expression of other enzymes such as epoxide hydrolase and phase II enzymes may determine the detoxification or metabolic activation of BaP.

  16. Inhibition of aryl hydrocarbon receptor transactivation and DNA adduct formation by CYP1 isoform-selective metabolic deactivation of benzo[a]pyrene.

    PubMed

    Endo, Kaori; Uno, Shigeyuki; Seki, Taiichiro; Ariga, Toyohiko; Kusumi, Yoshiaki; Mitsumata, Masako; Yamada, Sachiko; Makishima, Makoto

    2008-07-15

    Benzo[a]pyrene (BaP), a polyaromatic hydrocarbon produced by the combustion of cigarettes and coke ovens, is a known procarcinogen. BaP activates the aryl hydrocarbon receptor (AhR) and induces the expression of a battery of genes, including CYP1A1, which metabolize BaP to toxic compounds. The possible role of CYP1 enzymes in mediating BaP detoxification or metabolic activation remains to be elucidated. In this study, we assessed the effects of CYP1 enzymes (CYP1A1, CYP1A2 and CYP1B1) on BaP-induced AhR transactivation and DNA adduct formation in HEK293 cells and HepG2 cells. Transfection of CYP1A1 and CYP1B1, but not CYP1A2, suppressed BaP-induced activation of AhR. Expression of CYP1A1 and CYP1A2, but not CYP1B1, inhibited DNA adduct formation in BaP-treated HepG2 cells. These results indicate that CYP1A1 and CYP1B1 play a role in deactivation of BaP on AhR and that CYP1A1 and CYP1A2 are involved in BaP detoxification by suppressing DNA adduct formation. BaP treatment did not induce DNA adduct formation in HEK293 cells, even after transfection of CYP1 enzymes, suggesting that expression of CYP1 enzymes is not sufficient for DNA adduct formation. Lower expression of epoxide hydrolase and higher expression of glutathione S-transferase P1 (GSTP1) and GSTM1/M2 were observed in HEK293 cells compared with HepG2 cells. Dynamic expression of CYP1A1, CYP1A2 and CYP1B1 along with expression of other enzymes such as epoxide hydrolase and phase II enzymes may determine the detoxification or metabolic activation of BaP.

  17. Peptide hydrolase activities of the mucosa of human small intestine

    PubMed Central

    Heizer, William D.; Laster, Leonard

    1969-01-01

    Few studies have been published on peptide hydrolase activities of human small intestine mucosa. We developed methods to screen tissue extracts for such enzymes and to quantitate hydrolase activities for dipeptides containing the aromatic amino acid L-phenylalanine. The screening procedure indicated glycyl-L-proline hydrolase activity was reduced in biopsy specimens from patients with flattened intestinal mucosa. To explore this further, we established optimal assay conditions for hydrolase activities (a) glycyl-L-proline, (b) L-phenylalanyl-L-proline, (c) L-alanyl-L-phenylalanine, and (d) L-phenylalanylglycine. Biopsy specimens from patients with various intestinal disorders, but without flattened mucosa, and from three patients with flattened mucosa, showed a disproportionate reduction in activities (a) and (b), with the reduction being significantly more marked in the latter patients. We suggest that intestinal imidopeptide hydrolase activities, such as (a) and (b), are sensitive to changes in intestinal disease generally, particularly to the altered physiology associated with flattening of the mucosa, and are secondary to, rather than a cause of, the intestinal pathology. Our finding that intestinal alkaline phosphatase activity tended to parallel imidopeptide hydrolase activity, and that activity (a) was partially localized to the particulate fraction of mucosal homogenate, suggested that imidopeptide hydrolase activities may be located in the microvilli of the intestinal epithelium and that, like alkaline phosphatase activity, they may be reduced in flattened mucosae, in part at least because of the pathologic changes in the microvilli. In our studies of control subjects we did not detect peptide hydrolase activity deficiency analogous to asymptomatic disaccharidase deficiency. Images PMID:5765024

  18. Direct observation of enantiomer discrimination of epoxides by chiral salen complexes using ENDOR.

    PubMed

    Fallis, Ian A; Murphy, Damien M; Willock, David J; Tucker, Richard J; Farley, Robert D; Jenkins, Robert; Strevens, Robert R

    2004-12-08

    Electron nuclear double resonance (ENDOR) spectroscopy was used to investigate the weak enantioselective binding between chiral salen complexes [VO(1)] ((R,R)- and (S,S)-vanadyl N,N'-bis(3,5-di-tert-butylsalcylidene)-1,2-cyclohexanediamine) and chiral epoxides (e.g., (R)-/(S)-propylene epoxide, 5) in frozen (10 K) solution. Differences in epoxide binding by enatiomers of [VO(1)] was evidenced by changes to the 1H epoxide derived peaks in the ENDOR spectra, such that (R,R)-[VO(1)] + (R)-5 and (R,R)-[VO(1)] + (S)-5 yield noticeably different spectra. These changes were assigned to the small structural differences between the diastereomeric metal-epoxide adducts. Simulation of the spectra revealed differences in the VO...1Hepoxide distances for the diastereomeric pairs, which was confirmed by a complementary set of density functional theory (DFT) calculations. While the epoxide molecule is very weakly coordinated, ENDOR measurements of the racemic complex in racemic epoxide nevertheless indicated the preferential coordination of the (R)-5 to (R,R)-[VO(1)] (likewise (S)-(5) to (S,S)-[VO(1)]), which is favored over the binding of (S)-5 epoxide to (R,R)-[VO(1)] (and likewise (R)-5 epoxide to (S,S)-[VO(1)]). This demonstrates the unique power of the ENDOR technique to resolve weak chiral interactions for which EPR spectroscopy alone lacks sufficient resolution.

  19. Nickel-Catalyzed Regiodivergent Opening of Epoxides with Aryl Halides: Co-Catalysis Controls Regioselectivity

    PubMed Central

    Zhao, Yang; Weix, Daniel J.

    2014-01-01

    Epoxides are versatile intermediates in organic synthesis, but have rarely been employed in cross-coupling reactions. We report that bipyridine-ligated nickel can mediate the addition of functionalized aryl halides, a vinyl halide, and a vinyl triflate to epoxides under reducing conditions. For terminal epoxides, the regioselectivity of the reaction depends upon the co-catalyst employed. Iodide co-catalysis results in opening at the less hindered position via an iodohydrin intermediate. Titanocene co-catalysis results in opening at the more hindered position, presumably via TiIII-mediated radical generation. 1,2-Disubstituted epoxides are opened under both conditions to form predominantly the trans product. PMID:24341892

  20. Human adipose triglyceride lipase (PNPLA2) is not regulated by obesity and exhibits low in vitro triglyceride hydrolase activity.

    PubMed

    Mairal, A; Langin, D; Arner, P; Hoffstedt, J

    2006-07-01

    The recent identification of murine adipose triglyceride lipase (ATGL, now known as patatin-like phospholipase domain containing 2 [PNPLA2]), gene product of Pnpla2, has questioned the unique role of hormone sensitive lipase (HSL, now known as LIPE), gene product of Lipe, in fat cell lipolysis. Here, we investigated human ATGL and HSL adipose tissue gene expression and in vitro lipase activity. Levels of mRNA in adipose tissue from healthy obese and non-obese subjects were measured and lipase activity and adipocyte lipolytic capacity determined. HSL and ATGL cDNAs were transfected into Cos-7 cells and the relative tri- and diglyceride hydrolase activities were measured. Obesity was associated with a decreased subcutaneous and increased omental adipose tissue level of HSL mRNA. Subcutaneous HSL mRNA content was normalised upon weight reduction. In contrast, ATGL mRNA levels were unaffected by obesity and weight reduction. A high adipose tissue lipase activity was associated with increased maximal lipolysis and increased HSL, but not with ATGL mRNA levels. The in vitro triglyceride hydrolase activity of HSL was markedly higher than that of ATGL and contrary to HSL, ATGL was devoid of diglyceride hydrolase activity. The use of a selective HSL-inhibitor resulted in complete inhibition of HSL-mediated tri- and diglyceride hydrolase activity. The pH profile of human white adipose tissue triolein hydrolase activity was identical to that of HSL but differed from the ATGL profile. HSL, but not ATGL gene expression shows a regulation according to obesity status and is associated with increased adipose tissue lipase activity. Moreover, HSL has a higher capacity than ATGL to hydrolyse triglycerides in vitro.

  1. Leukotriene A4 hydrolase: Selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375

    PubMed Central

    Rudberg, Peter C.; Tholander, Fredrik; Thunnissen, Marjolein M. G. M.; Samuelsson, Bengt; Haeggström, Jesper Z.

    2002-01-01

    Leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) hydrolase (LTA4H)/aminopeptidase is a bifunctional zinc metalloenzyme that catalyzes the final and rate-limiting step in the biosynthesis of leukotriene B4 (LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a classical chemoattractant and immune modulating lipid mediator. Two chemical features are key to the bioactivity of LTB4, namely, the chirality of the 12R-hydroxyl group and the cis-trans-trans geometry of the conjugated triene structure. From the crystal structure of LTA4H, a hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 was identified in a narrow and otherwise hydrophobic pocket, believed to bind LTA4. In addition, Asp-375 belongs to peptide K21, a previously characterized 21-residue active site-peptide to which LTA4 binds during suicide inactivation. In the present report we used site-directed mutagenesis and x-ray crystallography to show that Asp-375, but none of the other candidate residues, is specifically required for the epoxide hydrolase activity of LTA4H. Thus, mutation of Asp-375 leads to a selective loss of the enzyme's ability to generate LTB4 whereas the aminopeptidase activity is preserved. We propose that Asp-375, possibly assisted by Gln-134, acts as a critical determinant for the stereoselective introduction of the 12R-hydroxyl group and thus the biological activity of LTB4. PMID:11917124

  2. Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

    SciTech Connect

    Vose, Sarah C.; Holland, Nina T.; Eskenazi, Brenda; Casida, John E.

    2007-10-01

    Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC{sub 50} values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined.

  3. Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration[S

    PubMed Central

    Jin, Jing; Zheng, Yuxiang; Boeglin, William E.; Brash, Alan R.

    2013-01-01

    Leukotriene (LT)A4 and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA4 is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A4. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA4 and converts 5R-HPETE to the cis epoxide 5-epi-LTA4, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations. PMID:23242647

  4. Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates.

    PubMed Central

    Alegret, M.; Ferrando, R.; Vázquez, M.; Adzet, T.; Merlos, M.; Laguna, J. C.

    1994-01-01

    1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7915611

  5. Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants.

    PubMed

    Nishikawa, Kaori; Li, Hang; Kawamura, Ryoichi; Osaka, Hitoshi; Wang, Yu-Lai; Hara, Yoko; Hirokawa, Takatsugu; Manago, Yoshimasa; Amano, Taiju; Noda, Mami; Aoki, Shunsuke; Wada, Keiji

    2003-04-25

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.

  6. Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

    SciTech Connect

    Chow, S.A.; Falany, J.L.; Fischer, L.J. )

    1989-06-01

    The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic beta-cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of 3H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.

  7. Investigation of the mechanism of phosphonoacetaldehyde hydrolase

    SciTech Connect

    Hepburn, T.W.; Olsen, D.B.; Dunaway-Mariano, D.; Mariano, P.S.

    1986-05-01

    The authors are presently studying enzymes which catalyze the formation and cleavage of carbon phosphorous bonds. In 1970 LaNauze et. al. reported the isolation of one enzyme of interest - phosphonoacetaldehyde hydrolase from a mutant of Bacillus cereus. This enzyme catalyzes the hydrolysis of phosphonoaldehyde to acetaldehyde and inorganic phosphate. They have isolated phosphonatase from wild type B. cereus (grown on 2-aminoethylphosphonate as the P/sub i/ source) and have used /sup 1/H-NMR and /sup 31/P-NMR techniques to determine the products of the enzyme reaction as phosphate and acetaldehyde. The mechanism of the enzyme could involve the formation of a Schiff base between phosphonoacetaldehyde and lysine or it might only require Mg/sup + +/, an essential cofactor for activity. To distinguish between these possibilities they have begun to look at the Schiff base formation in more detail. NaBH/sub 4/ was found to inactivate the enzyme in the presence of substrate but not in its absence. This is consistent with results obtained for the enzyme isolated from the mutant bacteria. In addition treatment of the wild type enzyme with tritiated NaBH/sub 4/ resulted in significant incorporation of radiolabel into the protein as compared to the control. These results tentatively suggest that hydrolysis proceeds via a covalent imine intermediate.

  8. Epoxyeicosatrienoic acids induce growth inhibition and calpain/caspase-12 dependent apoptosis in PDGF cultured 3T6 fibroblast.

    PubMed

    Nieves, Diana; Moreno, Juan J

    2007-11-01

    Previous studies have demonstrated that arachidonic acid (AA) metabolites released by the cyclooxygenase pathway is involved in serum-induced 3T6 fibroblast cycle progression and proliferation. However, these results also suggest that other AA cascade pathways might be involved. Recently, we also described the role of hydroxyeicosatetraenoic acids, which are produced by cytochrome P450 monooxygenases (CYP), in 3T6 fibroblast growth. AA can be also metabolized by the epoxygenase activity of CYP-producing epoxyeicosatrienoic acids (EETs). Finally, the cytosolic epoxide hydrolases catalyze the hydration of the EETs, transforming them into dihydroxyeicosatetraenoic acids (DHETEs). In this work, we have studied the role of the EETs/DHETEs on 3T6 fibroblasts growth. Our results show that PDGF stimulates 3T6 fibroblast proliferation and [3H]thymidine incorporation, while the addition of 5,6-EET, 8,9-EET, 11,12-EET or 14,15-EET (0.1-1 microM) inhibit these processes. Furthermore, 5,6-DHETE and 11,12-DHETE (0.1-1 microM) also inhibit cell proliferation