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Sample records for carboxylesterases

  1. CARBOXYLESTERASES IN ENANTIOSELECTIVE SYNTHESIS OF ORGANIC COMPOUNDS

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    E. A. Shesterenko

    2013-02-01

    Full Text Available The classification, structure, and mechanism of catalytic action of carboxylesterase of different origin are presented in the review. The prospects of carboxylesterases application for metabolism and both several drugs and prodrugs activation investigation in vitro are shown. The enzyme usage as biocatalyst of stereoselective hydrolysis and synthesis of a wide range of acyclic, carbocyclic and heterocyclic compounds — esters are also urgent. It was established that enantiomers obtainable with the help of carboxylesterase are characterized by high chemical yields and optical purity; immobilization on different supports stabilizes the enzyme and allows the repeated usage of obtained biocatalysts. The own studies conducted and the enzymatic hydrolysis features of news 3-acylhydroxy-1,4-benzodiasepin-2-ones — potential anxiolytic and hypnotic means, with a help of pig liver microsomal fraction carboxylesterase have been established. For the first time the enantioselective hydrolysis of 3-acetoxy-7-bromo-1-methyl-5-phenyl-1,2-dihydro-3H-1,4-benzdiazepine-2-one was accomplished using free and immobilized in phyllophorine and alginate, stabilized by Ca2+ microsomal fraction. The S-enantiomer of substrate was isolated, which suggests the increased specificity of pig liver microsomal fraction carboxylesterase to its R-enantiomer.

  2. Synthetic cannabimimetic agents metabolized by carboxylesterases

    DEFF Research Database (Denmark)

    Thomsen, Ragnar; Nielsen, Line M; Holm, Niels B

    2015-01-01

    , the metabolism of two indazole carboxamide derivatives, AB-PINACA and AB-FUBINACA, was investigated by using human liver microsomes (HLM). For both compounds, a major metabolic pathway was the enzymatic hydrolysis of the primary amide, resulting in the major metabolites AB-PINACA-COOH and AB-FUBINACA-COOH. Other...... major metabolic pathways were mono-hydroxylation of the N-pentyl chain in AB-PINACA and mono-hydroxylation of the 1-amino-3-methyl-1-oxobutane moiety in AB-FUBINACA. To identify the enzyme(s) responsible for the amide hydrolysis, incubations with recombinant carboxylesterases and human serum, as well...... in documenting drug usage in forensic and clinical screening. Additionally, the identification of CES1 as the main enzyme hydrolyzing these compounds improves our knowledge in the emerging field of xenobiotic metabolism by esterases....

  3. Carboxylesterase-mediated insecticide resistance: Quantitative increase induces broader metabolic resistance than qualitative change.

    Science.gov (United States)

    Cui, Feng; Li, Mei-Xia; Chang, Hai-Jing; Mao, Yun; Zhang, Han-Ying; Lu, Li-Xia; Yan, Shuai-Guo; Lang, Ming-Lin; Liu, Li; Qiao, Chuan-Ling

    2015-06-01

    Carboxylesterases are mainly involved in the mediation of metabolic resistance of many insects to organophosphate (OP) insecticides. Carboxylesterases underwent two divergent evolutionary events: (1) quantitative mechanism characterized by the overproduction of carboxylesterase protein; and (2) qualitative mechanism caused by changes in enzymatic properties because of mutation from glycine/alanine to aspartate at the 151 site (G/A151D) or from tryptophan to leucine at the 271 site (W271L), following the numbering of Drosophila melanogaster AChE. Qualitative mechanism has been observed in few species. However, whether this carboxylesterase mutation mechanism is prevalent in insects remains unclear. In this study, wild-type, G/A151D and W271L mutant carboxylesterases from Culex pipiens and Aphis gossypii were subjected to germline transformation and then transferred to D. melanogaster. These germlines were ubiquitously expressed as induced by tub-Gal4. In carboxylesterase activity assay, the introduced mutant carboxylesterase did not enhance the overall carboxylesterase activity of flies. This result indicated that G/A151D or W271L mutation disrupted the original activities of the enzyme. Less than 1.5-fold OP resistance was only observed in flies expressing A. gossypii mutant carboxylesterases compared with those expressing A. gossypii wild-type carboxylesterase. However, transgenic flies universally showed low resistance to OP insecticides compared with non-transgenic flies. The flies expressing A. gossypii W271L mutant esterase exhibited 1.5-fold resistance to deltamethrin, a pyrethroid insecticide compared with non-transgenic flies. The present transgenic Drosophila system potentially showed that a quantitative increase in carboxylesterases induced broader resistance of insects to insecticides than a qualitative change.

  4. Structure, function and applications of carboxylesterases from insects for insecticide resistance.

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    Yan, Shuaiguo; Cui, Feng; Qiao, Chuanling

    2009-01-01

    Carboxylesterases (EC 3.1.1.1) distribute broadly in insects, and play an important role in the metabolism with various functions. This paper reviews the insect carboxylesterases including the definitions and reaction mechanism, classification, structural context, functions especially on insecticide resistance, and its application.

  5. Effects of alcohol on human carboxylesterase drug metabolism

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    Parker, Robert B.; Hu, Zhe-Yi; Meibohm, Bernd; Laizure, S. Casey

    2015-01-01

    Background and Objective Human carboxylesterase-1 (CES1) and human carboxylesterase-2 (CES2) play an important role in metabolizing many medications. Alcohol is a known inhibitor of these enzymes but the relative effect on CES1 and CES2 is unknown. The aim of this study is to determine the impact of alcohol on the metabolism of specific probes for CES1 (oseltamivir) and CES2 (aspirin). Methods The effect of alcohol on CES1- and CES2-mediated probe drug hydrolysis was determined in vitro using recombinant human carboxylesterase. To characterize the in vivo effects of alcohol, healthy volunteers received each probe drug alone and in combination with alcohol followed by blood sample collection and determination of oseltamivir, aspirin, and respective metabolite pharmacokinetics. Results Alcohol significantly inhibited oseltamivir hydrolysis by CES1 in vitro but did not affect aspirin metabolism by CES2. Alcohol increased the oseltamivir area under the plasma concentration-time curve (AUC) from 0-6 h by 27% (range 11-46%, p=0.011) and decreased the metabolite/oseltamivir AUC 0-6 h ratio by 34% (range 25-41%, p<0.001). Aspirin pharmacokinetics were not affected by alcohol. Conclusions Alcohol significantly inhibited the hydrolysis of oseltamivir by CES1 both in vitro and in humans, but did not affect the hydrolysis of aspirin to salicylic acid by CES2. These results suggest that alcohol's inhibition of CES1 could potentially result in clinically significant drug interactions with other CES1-substrate drugs, but it is unlikely to significantly affect CES2-substrate drug hydrolysis. PMID:25511794

  6. In Vitro Drug Metabolism by Human Carboxylesterase 1

    DEFF Research Database (Denmark)

    Thomsen, Ragnar; Rasmussen, Henrik B; Linnet, Kristian

    2014-01-01

    Carboxylesterase 1 (CES1) is the major hydrolase in human liver. The enzyme is involved in the metabolism of several important therapeutic agents, drugs of abuse, and endogenous compounds. However, no studies have described the role of human CES1 in the activation of two commonly prescribed...... a panel of therapeutic drugs and drugs of abuse to assess their inhibition of the hydrolysis of p-nitrophenyl acetate by recombinant CES1 and human liver microsomes. The screening assay confirmed several known inhibitors of CES1 and identified two previously unreported inhibitors: the dihydropyridine...... calcium antagonist, isradipine, and the immunosuppressive agent, tacrolimus. CES1 plays a role in the metabolism of several drugs used in the treatment of common conditions, including hypertension, congestive heart failure, and diabetes mellitus; thus, there is a potential for clinically relevant drug-drug...

  7. Carboxylesterase activities toward pesticide esters in crops and weeds.

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    Gershater, Markus; Sharples, Kate; Edwards, Robert

    2006-12-01

    Proteins were extracted from maize, rice, sorghum, soybean, flax and lucerne; the weeds Abutilon theophrasti, Echinochloa crus-galli, Phalaris canariensis, Setaria faberii, Setaria viridis, Sorghum halepense and the model plant Arabidopsis thaliana and assayed for carboxylesterase activity toward a range of xenobiotics. These included the pro-herbicidal esters clodinafop-propargyl, fenoxaprop-ethyl, fenthioprop-ethyl, methyl-2,4-dichlorophenoxyacetic acid (2,4-d-methyl), bromoxynil-octanoate, the herbicide-safener cloquintocet-mexyl and the pyrethroid insecticide permethrin. Highest activities were recorded with alpha-naphthyl acetate and methylumbelliferyl acetate. Esters of p-nitrophenol were also readily hydrolysed, with turnover declining as the chain length of the acyl component increased. Activities determined with model substrates were much higher than those observed with pesticide esters and were of limited value in predicting the relative rates of hydrolysis of the crop protection agents. Substrate preferences with the herbicides were typically 2,4-d-methyl>clodinafop-propargyl>fenthioprop-ethyl, fenoxaprop-ethyl and bromoxynil-octanoate. Isoelectric focussing in conjunction with staining for esterase activity using alpha-naphthyl acetate as substrate confirmed the presence of multiple carboxylesterase isoenzymes in each plant, with major qualitative differences observed between species. The presence of serine hydrolases among the resolved isoenzymes was confirmed through their selective inhibition by the organophosphate insecticide paraoxon. Our studies identify potentially exploitable differences between crops and weeds in their ability to bioactivate herbicides by enzymic hydrolysis and also highlight the usefulness of Arabidopsis as a plant model to study xenobiotic biotransformation.

  8. Crystal Structure of the Geobacillus stearothermophilus Carboxylesterase Est55 and Its Activation of Prodrug CPT-11

    OpenAIRE

    Liu, Ping; Ewis, Hosam E.; Tai, Phang C.; Lu, Chung-Dar; Weber, Irene T.

    2006-01-01

    Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce SN-38, a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and 6.8 and resoluti...

  9. The roles of carboxylesterase and CYP isozymes on the in vitro metabolism of T-2 toxin

    Institute of Scientific and Technical Information of China (English)

    Ni-ni Lin; Jia Chen; Bin Xu; Xia Wei; Lei Guo; Jian-wei Xie

    2015-01-01

    Background: T-2 toxin poses a great threat to human health because it has the highest toxicity of the currently known trichothecene mycotoxins. To understand thein vivo toxicity and transformation mechanism of T-2 toxin, we investigated the role of two principal phaseⅠ drug-metabolizing enzymes (cytochrome P450 [CYP450] enzymes) on the metabolism of T-2 toxin, which are crucial to the metabolism of endogenous substances and xenobiotics. We also investigated carboxylesterase, which also plays an important role in the metabolism of toxic substances. Methods: A chemical inhibition method and a recombinant method were employed to investigate the metabolism of the T-2 toxin by the CYP450 enzymes, and a chemical inhibition method was used to study carboxylesterase metabolism. Samples incubated with human liver microsomes were analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC- QqQ MS) after a simple pretreatment. Results: In the presence of a carboxylesterase inhibitor, only 20% T-2 toxin was metabolized. When CYP enzyme inhibitors and a carboxylesterase inhibitor were both present, only 3% of the T-2 toxin was metabolized. The contributions of the CYP450 enzyme family to T-2 toxin metabolism followed the descending order CYP3A4, CYP2E1, CYP1A2, CYP2B6 or CYP2D6 or CYP2C19. Conclusions: Carboxylesterase and CYP450 enzymes are of great importance in T-2 toxin metabolism, in which carboxylesterase is predominant and CYP450 has a subordinate role. CYP3A4 is the principal member of the CYP450 enzyme family responsible for T-2 toxin metabolism. The metabolite produced by carboxylesterase is HT-2, and the metabolite produced by CYP 3A4 is 3’-OH T-2. The different metabolites show different toxicities. Our results will provide useful data concerning the toxic mechanism, the safety evaluation, and the health risk assessment of T-2 toxin.

  10. The role of human carboxylesterases in drug metabolism: have we overlooked their importance?

    Science.gov (United States)

    Laizure, S Casey; Herring, Vanessa; Hu, Zheyi; Witbrodt, Kevin; Parker, Robert B

    2013-02-01

    Carboxylesterases are a multigene family of mammalian enzymes widely distributed throughout the body that catalyze the hydrolysis of esters, amides, thioesters, and carbamates. In humans, two carboxylesterases, hCE1 and hCE2, are important mediators of drug metabolism. Both are expressed in the liver, but hCE1 greatly exceeds hCE2. In the intestine, only hCE2 is present and highly expressed. The most common drug substrates of these enzymes are ester prodrugs specifically designed to enhance oral bioavailability by hydrolysis to the active carboxylic acid after absorption from the gastrointestinal tract. Carboxylesterases also play an important role in the hydrolysis of some drugs to inactive metabolites. It has been widely believed that drugs undergoing hydrolysis by hCE1 and hCE2 are not subject to clinically significant alterations in their disposition, but evidence exists that genetic polymorphisms, drug-drug interactions, drug-disease interactions and other factors are important determinants of the variability in the therapeutic response to carboxylesterase-substrate drugs. The implications for drug therapy are far-reaching, as substrate drugs include numerous examples from widely prescribed therapeutic classes. Representative drugs include angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, antiplatelet drugs, statins, antivirals, and central nervous system agents. As research interest increases in the carboxylesterases, evidence is accumulating of their important role in drug metabolism and, therefore, the outcomes of pharmacotherapy.

  11. Enhancement of the enantioselectivity of carboxylesterase A by structure-based mutagenesis

    NARCIS (Netherlands)

    Godinho, Luis F.; Reis, Carlos R.; Rozeboom, Henriette J.; Dekker, Frank J.; Dijkstra, Bauke W.; Poelarends, Gerrit J.; Quax, Wim J.

    2012-01-01

    Previously studied Bacillus subtilis carboxylesterases (CesA and CesB) have potential for the kinetic resolution of racemic esters of 1,2-O-isopropylideneglycerol (IPG). CesA exhibits high activity but low enantioselectivity towards IPG-butyrate and IPG-caprylate, while the more enantioselective Ces

  12. Annotation and expression of carboxylesterases in the silkworm, Bombyx mori

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    Li Wen-Le

    2009-11-01

    Full Text Available Abstract Background Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed. Results Based on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics. Conclusion B. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded

  13. Human Carboxylesterase 2 Reverses Obesity-Induced Diacylglycerol Accumulation and Glucose Intolerance

    OpenAIRE

    Maxwell A. Ruby; Julie Massart; Hunerdosse, Devon M.; Milena Schönke; Jorge C. Correia; Louie, Sharon M.; Ruas, Jorge L.; Erik Näslund; Nomura, Daniel K.; Zierath, Juleen R.

    2017-01-01

    Serine hydrolases are a large family of multifunctional enzymes known to influence obesity. Here, we performed activity-based protein profiling to assess the functional level of serine hydrolases in liver biopsies from lean and obese humans in order to gain mechanistic insight into the pathophysiology of metabolic disease. We identified reduced hepatic activity of carboxylesterase 2 (CES2) and arylacetamide deacetylase (AADAC) in human obesity. In primary human hepatocytes, CES2 knockdown imp...

  14. Bacterial Expression and Kinetic Analysis of Carboxylesterase 001D from Helicoverpa armigera

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    Yongqiang Li

    2016-04-01

    Full Text Available Carboxylesterasesare an important class of detoxification enzymes involved in insecticide resistance in insects. A subgroup of Helicoverpa armigera esterases, known as Clade 001, was implicated in organophosphate and pyrethroid insecticide resistance due to their overabundance in resistant strains. In this work, a novel carboxylesterasegene 001D of H. armigera from China was cloned, which has an open reading frame of 1665 nucleotides encoding 554 amino acid residues. We used a series of fusion proteins to successfully express carboxylesterase 001D in Escherichia coli. Three different fusion proteins were generated and tested. The enzyme kinetic assay towards 1-naphthyl acetate showed all three purified fusion proteins are active with a Kcat between 0.35 and 2.29 s−1, and a Km between 7.61 and 19.72 μM. The HPLC assay showed all three purified fusion proteins had low but measurable hydrolase activity towards β-cypermethrin and fenvalerate insecticides (specific activities ranging from 0.13 to 0.67 μM·min−1·(μM−1·protein. The enzyme was stable up to 40 °C and at pH 6.0–11.0. The results imply that carboxylesterase 001D is involved in detoxification, and this moderate insecticide hydrolysis may suggest that overexpression of the gene to enhance insecticide sequestration is necessary to allow carboxylesterases to confer resistance to these insecticides in H. armigera.

  15. Structural and kinetic overview of the carboxylesterase EST2 from alicyclobacillus acidocaldarius: a comparison with the other members of the HSL family.

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    Mandrich, Luigi; Merone, Luigia; Manco, Giuseppe

    2009-01-01

    Thermophilic and hyperthermophilic carboxylesterases (EC 3.1.1.1) are excellent model systems for studying structure function relationships as well as in vitro and in vivo evolution and possible biotechnological applications. In this paper we review the main aspect of one of most studied microbial representative of the hormone sensitive lipase family (HSL), namely carboxylesterase 2 (EST2) from Alicyclobacillus acidocaldarius.

  16. Correlation between carboxylesterase alleles and insecticide resistance in Culex pipiens complex from China

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    Liu Yangyang

    2011-12-01

    Full Text Available Abstract Background In China, large amounts of chemical insecticides are applied in fields or indoors every year, directly or indirectly bringing selection pressure on vector mosquitoes. Culex pipiens complex has evolved to be resistant to all types of chemical insecticides, especially organophosphates, through carboxylesterases. Six resistant carboxylesterase alleles (Ester were recorded previously and sometimes co-existed in one field population, representing a complex situation for the evolution of Ester genes. Results In order to explore the evolutionary scenario, we analyzed the data from an historical record in 2003 and a recent investigation on five Culex pipiens pallens populations sampled from north China in 2010. Insecticide bioassays showed that these five populations had high resistance to pyrethroids, medium resistance to organophosphates, and low resistance to carbamates. Six types of Ester alleles, EsterB1, Ester2, Ester8, Ester9, EsterB10, and Ester11 were identified, and the overall pattern of their frequencies in geographic distribution was consistent with the report seven years prior to this study. Statistical correlation analysis indicated that Ester8 and Ester9 positively correlated with resistance to four insecticides, and EsterB10 to one insecticide. The occurrences of these three alleles were positively correlated, while the occurrence of EsterB1 was negatively correlated with Ester8, indicating an allelic competition. Conclusion Our analysis suggests that one insecticide can select multiple Ester alleles and one Ester allele can work on multiple insecticides. The evolutionary scenario of carboxylesterases under insecticide selection is possibly "one to many".

  17. Crystal Structure of the Geobacillus stearothermophilus Carboxylesterase Est55 and Its Activation of Prodrug CPT-11

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    Liu, Ping; Ewis, Hosam E.; Tai, Phang C.; Lu, Chung-Dar; Weber, Irene T.

    2007-01-01

    Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce SN-38, a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and 6.8 and resolution of 2.0 and 1.58 Å, respectively. Est55 folds into three domains, a catalytic domain, an α/β domain and a regulatory domain. The structure is in an inactive form; the side chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy. PMID:17239398

  18. Demonstration of carboxylesterase in cytology samples of human nasal respiratory epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Rodgers, D.A.; Nikula, K.J.; Avila, K. [and others

    1995-12-01

    The epithelial lining of the nasal airways is a target for responses induced by a variety of toxicant exposures. The high metabolic capacity of this tissue has been suggested to play a role in both protection of the airways through detoxication of certain toxicants, as well as in activation of other compounds to more toxic metabolites. Specifically, nasal carboxylesterase (CE) has been shown to mediate the toxicity of inhaled esters and acrylates by converting them to more toxic acid and alcohol metabolites which can be cytotoxic and/or carcinogenic to the nasal mucosa. Due to difficulties in extrapolating rodent models to human, new paradigms using human cells and tissues are essential to understanding and evaluating the metabolic processes in human nasal epithelium.

  19. Mammalian carboxylesterase (CES) releases GPI-anchored proteins from the cell surface upon lipid raft fluidization.

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    Orihashi, Kaoru; Tojo, Hiromasa; Okawa, Katsuya; Tashima, Yuko; Morita, Takashi; Kondoh, Gen

    2012-03-01

    Mammalian carboxylesterase (CES) is well known as a biotransformation enzyme for prodrugs and xenobiotics. Here, we purified CES as a GPI-anchored protein (GPI-AP)-releasing factor (GPIase) that releases such protein from the cell surface. All five isoforms of CES showed this activity to various degrees. When the serine residue of the catalytic triad for esterase was replaced by alanine, esterase activity was completely disrupted, while full GPIase activity remained, suggesting that these two activities are exhibited via different mechanisms. CES6, a new class of mammalian CES, exhibited the highest GPIase activity and released specific GPI-APs from the cell surface after lipid raft fluidization. The released product contained a GPI component, indicating that GPI-AP was released by cleavage in GPI. These results revealed for the first time that CES recognizes and catalyzes macromolecule GPI-AP as well as small molecules.

  20. A Conserved Carboxylesterase Is a SUPPRESSOR OF AVRBST-ELICITED RESISTANCE in Arabidopsis[W][OA

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    Cunnac, Sébastien; Wilson, Ariane; Nuwer, Jamie; Kirik, Angela; Baranage, Gayathri; Mudgett, Mary Beth

    2007-01-01

    AvrBsT is a type III effector from Xanthomonas campestris pv vesicatoria that is translocated into plant cells during infection. AvrBsT is predicted to encode a Cys protease that targets intracellular host proteins. To dissect AvrBsT function and recognition in Arabidopsis thaliana, 71 ecotypes were screened to identify lines that elicit an AvrBsT-dependent hypersensitive response (HR) after Xanthomonas campestris pv campestris (Xcc) infection. The HR was observed only in the Pi-0 ecotype infected with Xcc strain 8004 expressing AvrBsT. To create a robust pathosystem to study AvrBsT immunity in Arabidopsis, the foliar pathogen Pseudomonas syringae pv tomato (Pst) strain DC3000 was engineered to translocate AvrBsT into Arabidopsis by the Pseudomonas type III secretion (T3S) system. Pi-0 leaves infected with Pst DC3000 expressing a Pst T3S signal fused to AvrBsT-HA (AvrBsTHYB-HA) elicited HR and limited pathogen growth, confirming that the HR leads to defense. Resistance in Pi-0 is caused by a recessive mutation predicted to inactivate a carboxylesterase known to hydrolyze lysophospholipids and acylated proteins in eukaryotes. Transgenic Pi-0 plants expressing the wild-type Columbia allele are susceptible to Pst DC3000 AvrBsTHYB-HA infection. Furthermore, wild-type recombinant protein cleaves synthetic p-nitrophenyl ester substrates in vitro. These data indicate that the carboxylesterase inhibits AvrBsT-triggered phenotypes in Arabidopsis. Here, we present the cloning and characterization of the SUPPRESSOR OF AVRBST-ELICITED RESISTANCE1. PMID:17293566

  1. A conserved carboxylesterase is a SUPPRESSOR OF AVRBST-ELICITED RESISTANCE in Arabidopsis.

    Science.gov (United States)

    Cunnac, Sébastien; Wilson, Ariane; Nuwer, Jamie; Kirik, Angela; Baranage, Gayathri; Mudgett, Mary Beth

    2007-02-01

    AvrBsT is a type III effector from Xanthomonas campestris pv vesicatoria that is translocated into plant cells during infection. AvrBsT is predicted to encode a Cys protease that targets intracellular host proteins. To dissect AvrBsT function and recognition in Arabidopsis thaliana, 71 ecotypes were screened to identify lines that elicit an AvrBsT-dependent hypersensitive response (HR) after Xanthomonas campestris pv campestris (Xcc) infection. The HR was observed only in the Pi-0 ecotype infected with Xcc strain 8004 expressing AvrBsT. To create a robust pathosystem to study AvrBsT immunity in Arabidopsis, the foliar pathogen Pseudomonas syringae pv tomato (Pst) strain DC3000 was engineered to translocate AvrBsT into Arabidopsis by the Pseudomonas type III secretion (T3S) system. Pi-0 leaves infected with Pst DC3000 expressing a Pst T3S signal fused to AvrBsT-HA (AvrBsTHYB-HA) elicited HR and limited pathogen growth, confirming that the HR leads to defense. Resistance in Pi-0 is caused by a recessive mutation predicted to inactivate a carboxylesterase known to hydrolyze lysophospholipids and acylated proteins in eukaryotes. Transgenic Pi-0 plants expressing the wild-type Columbia allele are susceptible to Pst DC3000 AvrBsTHYB-HA infection. Furthermore, wild-type recombinant protein cleaves synthetic p-nitrophenyl ester substrates in vitro. These data indicate that the carboxylesterase inhibits AvrBsT-triggered phenotypes in Arabidopsis. Here, we present the cloning and characterization of the SUPPRESSOR OF AVRBST-ELICITED RESISTANCE1.

  2. Identification of carboxylesterase genes implicated in temephos resistance in the dengue vector Aedes aegypti.

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    Rodolphe Poupardin

    2014-03-01

    Full Text Available BACKGROUND: Thailand is currently experiencing one of its worst dengue outbreaks in decades. As in most countries where this disease is endemic, dengue control in Thailand is largely reliant on the use of insecticides targeting both immature and adult stages of the Aedes mosquito, with the organophosphate insecticide, temephos, being the insecticide of choice for attacking the mosquito larvae. Resistance to temephos was first detected in Aedes aegypti larvae in Thailand approximately 25 years ago but the mechanism responsible for this resistance has not been determined. PRINCIPAL FINDINGS: Bioassays on Ae. aegypti larvae from Thailand detected temephos resistance ratios ranging from 3.5 fold in Chiang Mai to nearly 10 fold in Nakhon Sawan (NS province. Synergist and biochemical assays suggested a role for increased carboxylesterase (CCE activities in conferring temephos resistance in the NS population and microarray analysis revealed that the CCE gene, CCEae3a, was upregulated more than 60 fold in the NS population compared to the susceptible population. Upregulation of CCEae3a was shown to be partially due to gene duplication. Another CCE gene, CCEae6a, was also highly regulated in both comparisons. Sequencing and in silico structure prediction of CCEae3a showed that several amino acid polymorphisms in the NS population may also play a role in the increased resistance phenotype. SIGNIFICANCE: Carboxylesterases have previously been implicated in conferring temephos resistance in Ae aegypti but the specific member(s of this family responsible for this phenotype have not been identified. The identification of a strong candidate is an important step in the development of new molecular diagnostic tools for management of temephos resistant populations and thus improved control of dengue.

  3. Tissue distribution, isozyme abundance and sensitivity to chlorpyrifos-oxon of carboxylesterases in the earthworm Lumbricus terrestris

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    Sanchez-Hernandez, Juan C. [Laboratory of Ecotoxicology, Faculty of Environmental Science, University of Castilla-La Mancha, Avda. Carlos III, 45071 Toledo (Spain)], E-mail: juancarlos.sanchez@uclm.es; Wheelock, Craig E. [Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 171 77, Stockholm (Sweden)

    2009-01-15

    A laboratory-based study was conducted to determine the basal carboxylesterase (CbE) activity in different tissues of the earthworm Lumbricus terrestris, and its sensitivity to the organophosphate (OP) pesticide chlorpyrifos-oxon (CPx). Carboxylesterase activity was found in the pharynx, crop, gizzard, anterior intestine, wall muscle and reproductive tissues of L. terrestris, and multiple tissue-specific isozymes were observed by native gel electrophoresis. Esterase activity and sensitivity to CPx inhibition varied on a tissue- and substrate-specific basis, suggesting isoforms-specific selectivity to OP-mediated inhibition. Three practical issues are recommended for the use of earthworm CbE activity as a biomarker of pesticide exposure: (i) CbE should be measured using several routine substrates, (ii) it should be determined in selected tissues instead of whole organism homogenate, and (iii) earthworm CbE activity should be used in conjuncture with other common biomarkers (e.g., ChE) within a multibiomarker approach to assess field exposure of OPs, and potentially other agrochemicals. - The measurement of carboxylesterase inhibition in earthworm is a sensitive and complementary biomarker of pesticide exposure.

  4. Quantitative and qualitative changes of the carboxylesterase associated with beta-cypermethrin resistance in the housefly, Musca domestica (Diptera: Muscidae).

    Science.gov (United States)

    Zhang, Lan; Shi, Jing; Shi, Xueyan; Liang, Pei; Gao, Junping; Gao, Xiwu

    2010-05-01

    Mechanisms of esterase-mediated pyrethroid resistance were analyzed based on our previous works in a strain of the housefly, Musca domestica. The carboxylesterase gene, MdalphaE7, was cloned and sequenced from susceptible (CSS) and resistant (CRR) strains, and a total of nine amino acid substitutions were found. The mutation, Trp(251)-Ser appeared to play a role in beta-cypermethrin resistance and cross-resistance between organophosphates (OPs) and pyrethroids in the CRR strain. Quantitative real-time PCR showed that MdalphaE7 was over-expressed in the CRR strain, the reciprocal cross progeny F(1) and back-cross progeny BC(2) compared with the CSS strain, respectively. Two alpha-cynaoester substrates as surrogates for beta-cypermethrin and deltamethrin, were synthesized to determine the pyrethroid hydrolase activity. Results showed that carboxylesterases from the CRR strain hydrolyzed cypermethrin/deltamethrin-like substrate 9.05- and 13.53-fold more efficiently than those from the CSS strain, respectively. Our studies suggested that quantitative and qualitative changes in the carboxylesterase might contribute together to pyrethroid resistance in the CRR strain.

  5. Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases.

    Science.gov (United States)

    Chahinian, Henri; Ali, Yassine Ben; Abousalham, Abdelkarim; Petry, Stefan; Mandrich, Luigi; Manco, Guiseppe; Canaan, Stephane; Sarda, Louis

    2005-12-30

    We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.

  6. A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues.

    Science.gov (United States)

    Jin, Qiang; Feng, Lei; Wang, Dan-Dan; Dai, Zi-Ru; Wang, Ping; Zou, Li-Wei; Liu, Zhi-Hong; Wang, Jia-Yue; Yu, Yang; Ge, Guang-Bo; Cui, Jing-Nan; Yang, Ling

    2015-12-30

    In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2) based on the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1,8-naphthalimide (NAH), which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity, and has been successfully applied to determine the real activities of hCE2 in complex biological samples such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bioimaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems.

  7. Testing the evolvability of an insect carboxylesterase for the detoxification of synthetic pyrethroid insecticides.

    Science.gov (United States)

    Coppin, Chris W; Jackson, Colin J; Sutherland, Tara; Hart, Peter J; Devonshire, Alan L; Russell, Robyn J; Oakeshott, John G

    2012-05-01

    Esterases have been implicated in metabolic resistance to synthetic pyrethroids in several insect species but little is yet known of the molecular basis for these effects. In this work modern directed evolution technology was used to test to what extent it is possible to genetically enhance the pyrethroid hydrolytic activity of the E3 carboxylesterase from the blowfly Lucilia cuprina. High throughput screening of a random mutant library with individual stereoisomers of fluorogenic analogues of two type II pyrethroids identified 17 promising variants that were then also tested with the commercial pyrethroid deltamethrin. Between them, these variants displayed significantly improved activities for all the substrates tested. Amino acid substitutions at ten different residues were clearly implicated in the improvements, although most only enhanced activity for a subset of the stereoisomers. Several new combinations of the most promising amino acid substitutions were then made, and negative epistatic effects were found in most of the combinations, but significant improvements were also found in a minority of them. The best mutant recovered contained three amino acid changes and hydrolysed deltamethrin at more than 100 times the rate of wild-type E3. Structural analysis shows that nine of the ten mutated residues improving pyrethroid or analogue activities cluster in putative substrate binding pockets in the active site, with the three mutations of largest effect all increasing the volume of the acyl pocket.

  8. Characterization of Carboxylesterase Associated with Malathion Insensitivity in the Field Population of the Oriental Migratory Locust

    Institute of Scientific and Technical Information of China (English)

    YANG Mei-ling; ZHANG Jian-zhen; ZHANG Jian-qin; GUO Ya-ping; MA En-bo

    2008-01-01

    Carboxylesterases (CarEs) from two field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), were examined to try to understand their contribution to malathion insensitivity. The CarEs activities in Wudi population (WD) were 1.75- and 1.50-fold significantly higher than those in Huangliu population (HL) when α-naphthyl acetate (α-NA) and β-naphthyl acetate were used as substrates, respectively. Such elevated CarEs activities presented in the WD could be because of an increased staining intensity of the α-NA-hydrolyzing CarEs as shown on the nondenaturing polyacrylamide gel electrophoresis. Inhibition studies of CarEs using paraoxon and malaoxon indicated that CarE activities in the HL were more strongly inhibited than those in the WD. Furthermore, a 449-bp DNA fragment of Care was obtained from L. migratoria manilensis. Hemiquantity reverse transcription-polymerase chain reaction analysis showed that CarE gene expression level in the WD was higher than that in the HL. The higher CarE activities and the increased CarE mRNA level in the WD appeared to be associated with decreased susceptibility to malathion in the WD due to the application of organophosphorus insecticides.

  9. Overexpression of carboxylesterase contributes to the attenuation of cyanotoxin microcystin-LR toxicity.

    Science.gov (United States)

    Takumi, Shota; Shimono, Tai; Ikema, Satoshi; Hotta, Yuki; Chigwechokha, Petros K; Shiozaki, Kazuhiro; Sugiyama, Yasumasa; Hashimoto, Mitsuru; Furukawa, Tatsuhiko; Komatsu, Masaharu

    2017-04-01

    Microcystin-LR is a hepatotoxin produced by several cyanobacteria. Its toxicity is mainly due to a inhibition of protein phosphatase, PP1 and PP2A. Previously, we used a cell line stably expressing uptake transporter for microcystin-LR, OATP1B3 (HEK293-OATP1B3 cells). In this study, to determine whether overexpression of carboxylesterase (CES), which degrades ester-group and amide-group, attenuates the cytotoxicity of microcystin-LR, we generated the HEK293-OATP1B3/CES2 double-transfected cells. HEK293-OATP1B3/CES2 cells showed high hydrolysis activity of p-nitrophenyl acetate (PNPA), which is an authentic substrate for esterase. CES activity in HEK293-OATP1B3/CES2 cells was approximately 3-fold higher than that in the HEK293-OATP1B3 cells. HEK293-OATP1B3/CES2 cells (IC50: 25.4±7.7nM) showed approximately 2.1-fold resistance to microcystin-LR than HEK293-OATP1B3 cells (IC50: 12.0±1.5nM). Moreover, the CES inhibition assay and microcystin-agarose pull down assay showed the possibility of the interaction between CES2 and microcystin-LR. Our results indicated that the overexpression of CES2 attenuates the cytotoxicity of microcystin-LR via interaction with microcystin-LR.

  10. An in vitro screening with emerging contaminants reveals inhibition of carboxylesterase activity in aquatic organisms.

    Science.gov (United States)

    Solé, Montserrat; Sanchez-Hernandez, Juan C

    2015-12-01

    Pharmaceuticals and personal care products (PPCPs) form part of the new generation of pollutants present in many freshwater and marine ecosystems. Although environmental concentrations of these bioactive substances are low, they cause sublethal effects (e.g., enzyme inhibition) in non-target organisms. However, little is known on metabolism of PPCPs by non-mammal species. Herein, an in vitro enzyme trial was performed to explore sensitivity of carboxylesterase (CE) activity of aquatic organisms to fourteen PPCPs. The esterase activity was determined in the liver of Mediterranean freshwater fish (Barbus meridionalis and Squalius laietanus), coastal marine fish (Dicentrarchus labrax and Solea solea), middle-slope fish (Trachyrhynchus scabrus), deep-sea fish (Alepocephalus rostratus and Cataetix laticeps), and in the digestive gland of a decapod crustacean (Aristeus antennatus). Results showed that 100μM of the lipid regulators simvastatin and fenofibrate significantly inhibited (30-80% of controls) the CE activity of all target species. Among the personal care products, nonylphenol and triclosan were strong esterase inhibitors in most species (36-68% of controls). Comparison with literature data suggests that fish CE activity is as sensitive to inhibition by some PPCPs as that of mammals, although their basal activity levels are lower than in mammals. Pending further studies on the interaction between PPCPs and CE activity, we postulate that this enzyme may act as a molecular sink for certain PPCPs in a comparable way than that described for the organophosphorus pesticides.

  11. A Characterization of Carboxylesterases in Rat and Guinea Pig - Their Heterogeneity and Role in Detoxication of Organophosphorus Compounds

    Science.gov (United States)

    1993-09-01

    lipase (EC 3.1.1.23) 1221, in both 12. Berg T, Boman D and Segelen P0, Induction of EC and KC may serve to protect the liver by hydrolysis of...organophosphorus compounds. CarbE isoenzymes have been sepa- rated from lung, liver , plasma and small intestine of rat and guinea pig mainly by chromatofocusing...different tissues of rat and guinea pig 18 5.5 Heterogeneity of carboxylesterases in rat liver cells 20 6 CONCLUSIONS 20 References 21 Errata 27

  12. Carboxylesterase 1 gene duplication and mRNA expression in adipose tissue are linked to obesity and metabolic function

    DEFF Research Database (Denmark)

    Friedrichsen, Martin; Poulsen, Pernille; Wojtaszewski, Jørgen;

    2013-01-01

    involved in the control of mRNA expression. Here, we investigated mRNA expression level in adipose tissue and its association with measures of adiposity and metabolic function in a population of elderly twins. Furthermore, the heritability of mRNA expression level in adipose tissue and the effect of gene......CONTEXT AND AIMS: Carboxylesterase 1 (CES1) appears to play an important role in the control of the metabolism of triglycerides and cholesterol in adipocytes and other cell types including hepatocytes. Therefore, it is relevant to gain insights into the genetic versus non-genetic mechanisms...

  13. Functional analysis of carboxylesterase in human induced pluripotent stem cell-derived enterocytes.

    Science.gov (United States)

    Kabeya, Tomoki; Matsumura, Wakana; Iwao, Takahiro; Hosokawa, Masakiyo; Matsunaga, Tamihide

    2017-04-22

    Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

  14. Enhancement of the enantioselectivity of carboxylesterase A by structure-based mutagenesis.

    Science.gov (United States)

    Godinho, Luis F; Reis, Carlos R; Rozeboom, Henriëtte J; Dekker, Frank J; Dijkstra, Bauke W; Poelarends, Gerrit J; Quax, Wim J

    2012-03-31

    Previously studied Bacillus subtilis carboxylesterases (CesA and CesB) have potential for the kinetic resolution of racemic esters of 1,2-O-isopropylideneglycerol (IPG). CesA exhibits high activity but low enantioselectivity towards IPG-butyrate and IPG-caprylate, while the more enantioselective CesB does not process IPG-butyrate and exhibits several-fold lower activity than CesA towards IPG-caprylate. A sequence and structure comparison allowed us to identify active site residues that may cause the difference in (enantio)selectivities of CesA and CesB towards these IPG esters. This structure-based approach led to the identification of two active site residues in CesA (F166 and F182), as promising candidates for mutagenesis in order to enhance its enantioselectivity. Mutagenesis of positions 166 and 182 in CesA yielded novel variants with enhanced enantioselectivity and without significant loss of catalytic activity. For IPG-butyrate, a CesA double mutant F166V/F182C (ER=13) was generated showing a ∼13-fold increased enantioselectivity as compared to wild-type CesA (E=1). For IPG-caprylate, we designed a CesA double mutant F166V/F182Y (ER=9) displaying a ∼5-fold increased enantioselectivity as compared to the wild-type enzyme (ER=2). These findings, combined with the results of molecular docking experiments, demonstrate the importance of residues at positions 166 and 182 for the enantioselectivity of CesA, and may contribute to the development of efficient biocatalysts.

  15. RNA interference revealed the roles of two carboxylesterase genes in insecticide detoxification in Locusta migratoria.

    Science.gov (United States)

    Zhang, Jianqin; Li, Daqi; Ge, Pingting; Yang, Meiling; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2013-10-01

    Carboxylesterases (CarEs) play key roles in metabolism of specific hormones and detoxification of dietary and environmental xenobiotics in insects. We sequenced and characterized CarE cDNAs putatively derived from two different genes named LmCesA1 and LmCesA2 from the migratory locust, Locusta migratoria, one of the most important agricultural pests in the world. The full-length cDNAs of LmCesA1 (1892 bp) and LmCesA2 (1643 bp) encode 543 and 501 amino acid residues, respectively. The two deduced CarEs share a characteristic α/β-hydrolase structure, including a catalytic triad composed of Ser-Glu (Asp)-His and a consensus sequence GQSAG, which suggests that both CarEs are biologically active. Phylogenetic analysis grouped both LmCesA1 and LmCesA2 into clade A which has been suggested to be involved in dietary detoxification. Both transcripts were highly expressed in all the nymphal and adult stages, but only slightly expressed in eggs. Analyses of tissue-dependent expression and in situ hybridization revealed that both transcripts were primarily expressed in gastric caeca. RNA interference (RNAi) of LmCesA1 and LmCesA2 followed by a topical application of carbaryl or deltamethrin did not lead to a significantly increased mortality with either insecticide. However, RNAi of LmCesA1 and LmCesA2 increased insect mortalities by 20.9% and 14.5%, respectively, when chlorpyrifos was applied. These results suggest that these genes might not play a significant role in detoxification of carbaryl and deltamethrin but are most likely to be involved in detoxification of chlorpyrifos in L. migratoria.

  16. Soluble Expression in Escherichia Coli and Purification of Human Carboxylesterase 1

    Institute of Scientific and Technical Information of China (English)

    WANG Lei; TONG Jin-ying; CAO Peng-rong; PENG Xiao-ning; YI Yin-sha; LV Yuan

    2014-01-01

    Objective: To achieve an optimized method for soluble expression of human carboxylesterase 1 (hCE-1) in escherichia coil and purification by Ni2+-NTA agarose affinity chromatography, to get improved protein yield and purity for further development of hepatocellular carcinoma (HCC) diagnosis ELISA kits. Methods: The best antigen epitopes of hCE1 were predicted by comparing secondary structure, flexible regions, hydrophilicity, antigenic index surface probability of residues. Afterwards, pET-42a (+) with a His-tag and a GST-tag was applied to form recombinant plasmid pET-42a (+)/hCE1, which facilitated purification when using Ni2+-NTA agarose affinity chromatography. Protein quality was measured by SDS-PAGE and BCA protein assay. Western-blot identification was also performed to ensure the correct expression of hCE1 protein. Results: The residues from 500 to 567 near C-terminal of hCE1 protein were considered the best epitopes which exhibited high hydrophilicity and high surface probability and relatively flexible secondary structure and low homology compared with hCE2 and hCE3. His-hCE1 500-567 fusion protein was achieved by IPTG-inducted expression with an expected mass of 42 kDa. After purification, the final product was specially identified, which reached over 95%purity and more than 10 mg/L of microbial culture. In Western blot, the purified fusion protein was recognized by anti-hCE1 monoclonal antibody, along with previous sequencing validation, which demonstrated the correct preparation of soluble hCE1 protein. Conclusion: This is an efficacious and affordable strategy to generate fusion hCE1 of high quality in E coli, which facilitates preparation of hCE1 monoclonal antibody and further HCC diagnosis research.

  17. Effect of Cellular Location of Human Carboxylesterase 2 on CPT-11 Hydrolysis and Anticancer Activity.

    Directory of Open Access Journals (Sweden)

    Yuan-Ting Hsieh

    Full Text Available CPT-11 is an anticancer prodrug that is clinically used for the treatment of metastatic colorectal cancer. Hydrolysis of CPT-11 by human carboxylesterase 2 (CE2 generates SN-38, a topoisomerase I inhibitor that is the active anti-tumor agent. Expression of CE2 in cancer cells is under investigation for the tumor-localized activation of CPT-11. CE2 is normally expressed in the endoplasmic reticulum of cells but can be engineered to direct expression of active enzyme on the plasma membrane or as a secreted form. Although previous studies have investigated different locations of CE2 expression in cancer cells, it remains unclear if CE2 cellular location affects CPT-11 anticancer activity. In the present study, we directly compared the influence of CE2 cellular location on substrate hydrolysis and CPT-11 cytotoxicity. We linked expression of CE2 and enhanced green fluorescence protein (eGFP via a foot-and-mouth disease virus 2A (F2A peptide to facilitate fluorescence-activated cell sorting to achieve similar expression levels of ER-located, secreted or membrane-anchored CE2. Soluble CE2 was detected in the medium of cells that expressed secreted and membrane-anchored CE2, but not in cells that expressed ER-retained CE2. Cancer cells that expressed all three forms of CE2 were more sensitive to CPT-11 as compared to unmodified cancer cells, but the membrane-anchored and ER-retained forms of CE2 were consistently more effective than secreted CE2. We conclude that expression of CE2 in the ER or on the membrane of cancer cells is suitable for enhancing CPT-11 anticancer activity.

  18. Selective production of 1-monocaprin by porcine liver carboxylesterase-catalyzed esterification: Its enzyme kinetics and catalytic performance.

    Science.gov (United States)

    Park, Kyung-Min; Lee, Jong-Hyuk; Hong, Sung-Chul; Kwon, Chang Woo; Jo, Minje; Choi, Seung Jun; Kim, Keesung; Chang, Pahn-Shick

    2016-01-01

    Porcine liver carboxylesterase (PLE) belongs to carboxylesterase family (EC 3.1.1.1) as a serine-type esterase. The PLE-catalyzed esterification of capric acid with glycerol in reverse micelles was investigated on the catalytic performance and enzyme kinetics. The most suitable structure of reverse micelles was comprised of isooctane (reaction medium) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT, anionic surfactant) with 0.1 of R-value ([water]/[surfactant]) and 3.0 of G/F-value ([glycerol]/[fatty acid]) for the PLE-catalyzed esterification. In the aspect of regio-selectivity, the PLE mainly produced 1-monocaprin without any other products (di- and/or tricaprins of subsequent reactions). Furthermore, the degree of esterification at equilibrium state (after 4 h from the initiation) was 62.7% under the optimum conditions at pH 7.0 and 60 °C. Based on Hanes-Woolf plot, the apparent Km and Vmax values were calculated to be 16.44 mM and 38.91 μM/min/mg protein, respectively.

  19. Adenoviral vector-mediated expression of a gene encoding secreted, EpCAM-targeted carboxylesterase-2 sensitises colon cancer spheroids to CPT-11

    NARCIS (Netherlands)

    Oosterhoff, D; Overmeer, RM; de Graaf, M; van der Meulen, IH; Giaccone, G; van Beusechem, VW; Haisma, HJ; Pinedo, HM; Gerritsen, WR

    2005-01-01

    CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is an anticancer agent in use for the treatment of colon cancer. In order to be fully active, CPT-11 needs to be converted into SN-38 (7-ethyl-10-hydroxycamptothecin) by the enzyme carboxylesterase (CE). In human

  20. A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila

    Directory of Open Access Journals (Sweden)

    Chertemps Thomas

    2012-06-01

    Full Text Available Abstract Background Insects respond to the spatial and temporal dynamics of a pheromone plume, which implies not only a strong response to 'odor on', but also to 'odor off'. This requires mechanisms geared toward a fast signal termination. Several mechanisms may contribute to signal termination, among which odorant-degrading enzymes. These enzymes putatively play a role in signal dynamics by a rapid inactivation of odorants in the vicinity of the sensory receptors, although direct in vivo experimental evidences are lacking. Here we verified the role of an extracellular carboxylesterase, esterase-6 (Est-6, in the sensory physiological and behavioral dynamics of Drosophila melanogaster response to its pheromone, cis-vaccenyl acetate (cVA. Est-6 was previously linked to post-mating effects in the reproductive system of females. As Est-6 is also known to hydrolyze cVA in vitro and is expressed in the main olfactory organ, the antenna, we tested here its role in olfaction as a putative odorant-degrading enzyme. Results We first confirm that Est-6 is highly expressed in olfactory sensilla, including cVA-sensitive sensilla, and we show that expression is likely associated with non-neuronal cells. Our electrophysiological approaches show that the dynamics of olfactory receptor neuron (ORN responses is strongly influenced by Est-6, as in Est-6° null mutants (lacking the Est-6 gene cVA-sensitive ORN showed increased firing rate and prolonged activity in response to cVA. Est-6° mutant males had a lower threshold of behavioral response to cVA, as revealed by the analysis of two cVA-induced behaviors. In particular, mutant males exhibited a strong decrease of male-male courtship, in association with a delay in courtship initiation. Conclusions Our study presents evidence that Est-6 plays a role in the physiological and behavioral dynamics of sex pheromone response in Drosophila males and supports a role of Est-6 as an odorant-degrading enzyme (ODE in male

  1. Carboxylesterase-involved metabolism of di-n-butyl phthalate in pumpkin (Cucurbita moschata) seedlings.

    Science.gov (United States)

    Lin, Qingqi; Chen, Siyuan; Chao, Yuanqing; Huang, Xiongfei; Wang, Shizhong; Qiu, Rongliang

    2017-01-01

    Uptake and accumulation by plants is a significant pathway in the migration and transformation of phthalate esters (PAEs) in the environment. However, limited information is available on the mechanisms of PAE metabolism in plants. Here, we investigated the metabolism of di-n-butyl phthalate (DnBP), one of the most frequently detected PAEs, in pumpkin (Cucurbita moschata) seedlings via a series of hydroponic experiments with an initial concentration of 10 mg L(-1). DnBP hydrolysis occurred primarily in the root, and two of its metabolites, mono-n-butyl phthalate (MnBP) and phthalic acid (PA), were detected in all plant tissues. The MnBP concentration was an order of magnitude higher than that of PA in shoots, which indicated MnBP was more readily transported to the shoot than was PA because of the former's dual hydrophilic and lipophilic characteristics. More than 80% of MnBP and PA were located in the cell water-soluble component except that 96% of MnBP was distributed into the two solid cellular fractions (i.e., cell wall and organelles) at 96 h. A 13-20% and 29-54% increase of carboxylesterase (CXE) activity shown in time-dependent and concentration-dependent experiments, respectively, indicated the involvement of CXEs in plant metabolism of DnBP. The level of CXE activity in root subcellular fractions was in the order: the cell water-soluble component (88-94%) > cell wall (3-7%) > cell organelles (3-4%), suggesting that the cell water-soluble component is the dominant locus of CXE activity and also the domain of CXE-catalyzed hydrolysis of DnBP. The addition of triphenyl phosphate, a CXE inhibitor, led to 43-56% inhibition of CXE activity and 16-25% increase of DnBP content, which demonstrated the involvement of CXEs in plant metabolism of DnBP. This study contributes to our understanding of enzymitic mechanisms of PAE transformation in plants.

  2. The stability of methyl-, ethyl- and fluoroethylesters against carboxylesterases in vitro: there is no difference

    Energy Technology Data Exchange (ETDEWEB)

    Nics, Lukas [Department of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna (Austria); Department of Nutritional Sciences, University of Vienna, A-1090 Vienna (Austria); Haeusler, Daniela [Department of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna (Austria); Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, A-1090 Vienna (Austria); Wadsak, Wolfgang [Department of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna (Austria); Department of Inorganic Chemistry, University of Vienna, A-1090 Vienna (Austria); Wagner, Karl-Heinz [Department of Nutritional Sciences, University of Vienna, A-1090 Vienna (Austria); Dudczak, Robert; Kletter, Kurt [Department of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna (Austria); Mitterhauser, Markus, E-mail: markus.mitterhauser@meduniwien.ac.a [Department of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna (Austria); Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, A-1090 Vienna (Austria); Hospital Pharmacy of the General Hospital of Vienna, A-1090 Vienna (Austria)

    2011-01-15

    Introduction: Carboxylesterases (CES) play a very important role in the hydrophilic biotransformation of a huge number of structurally diverse drugs and especially play a leading part in the catabolic pathway of carboxylesters or thioesters. Hence, the aim of the present study was the comparison of the in vitro stability of methyl- and ethylesters with fluoroethylesters. Methods: We incubated methyl 3{beta}-(4-iodophenyl)tropane-2{beta}-carboxylate ({beta}-CIT)/2-fluoroethyl 3{beta}-(4-iodophenyl)tropane-2{beta}-carboxylate (FE-CIT), methyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MTO)/ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (ETO)/2-fluoroethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (FETO), ethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo-[f]imidazo[1,5-a]-[1,4] diazepine-3-carboxylate (FMZ)/2-fluoroethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo-[f]imidazo[1,5-a]-[1,4] diazepine-3-carboxylate (FFMZ), methyl 1-phenylethyl-4-(N-propanoylanilino)piperidine-4-carboxylate (CFN)/2-fluoroethyl 1-phenylethyl-4-(N-propanoylanilino)piperidine-4-carboxylate ((FE-CF)N) and methyl 2,4-diethyl-3-methylsulfanylcarbonyl-6-phenylpyridine-5-carboxylate [(Me){sup 2}-SUPPY]/2-fluorethyl 2,4-diethyl-3-ethylsulfanylcarbonyl-6-phenylpyridine-5-carboxylate (FE-SUPPY) under physiological conditions. The enzymatic reactions were stopped at different time points and analyzed by a standard protocol. Results: The Michaelis-Menten constants (K{sub M}) and limiting velocities (V{sub max}) are comparable. The statistical K{sub M} values were as follows: {beta}-CIT/FE-CIT, P>.05; MTO/FETO, P>.06; ETO/FETO, P>.09; FMZ/FFMZ, P>.05; CFN/ (FE-CFN), P>.9; (Me){sup 2}-SUPPY/FE-SUPPY, P>.07. Conclusion: We found no statistical difference in stability against CES in vitro. These findings support the strategy to translate C-11-methyl-/ethylesters into their longer-lived F-18-fluoroethyl analogues.

  3. Automated evaluation of pharmaceutically active ionic liquids' (eco)toxicity through the inhibition of human carboxylesterase and Vibrio fischeri.

    Science.gov (United States)

    Costa, Susana P F; Justina, Vanessa D; Bica, Katharina; Vasiloiu, Maria; Pinto, Paula C A G; Saraiva, M Lúcia M F S

    2014-01-30

    The toxicity of 16 pharmaceutical active ionic liquids (IL-APIs) was evaluated by automated approaches based on sequential injection analysis (SIA). The implemented bioassays were centered on the inhibition of human carboxylesterase 2 and Vibrio fischeri, in the presence of the tested compounds. The inhibitory effects were quantified by calculating the inhibitor concentration required to cause 50% of inhibition (EC50). The EC50 values demonstrated that the cetylpyridinium group was one of the most toxic cations and that the imidazolium group was the less toxic. The obtained results provide important information about the safety of the studied IL-APIs and their possible use as pharmaceutical drugs. The developed automated SIA methodologies are robust screening bioassays, and can be used as a generic tools to identify the (eco)toxicity of the structural elements of ILs, contributing to a sustainable development of drugs.

  4. Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents

    DEFF Research Database (Denmark)

    Shi, Yuping; Pan, Yingjie; Li, Bailin

    2013-01-01

    Hx with other known BioH proteins revealed interesting diversity in their sensitivity to ionic and nonionic detergents and organic solvents, and BioHx exhibited exceptional resistance to organic solvents, being the most tolerant one amongst all known BioH enzymes. This ascribed BioHx as a novel carboxylesterase...... with a strong potential in industrial applications. CONCLUSIONS: This study constituted the first investigation of a novel bioHx gene in a biotin biosynthetic gene cluster cloned from an environmental metagenome. The bioHx gene was successfully cloned, expressed and characterized. The results demonstrated...... that BioHx is a novel carboxylesterase, displaying distinct biochemical properties with strong application potential in industry. Our results also provided the evidence for the effectiveness of functional metagenomic approach for identifying novel bioH genes from complex ecosystem....

  5. Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

    Science.gov (United States)

    Igetei, Joseph E; Liddell, Susan; El-Faham, Marwa; Doenhoff, Michael J

    2016-04-01

    A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine β-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or β-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.

  6. Carboxylesterase-2 is a highly sensitive target of the antiobesity agent orlistat with profound implications in the activation of anticancer prodrugs.

    Science.gov (United States)

    Xiao, Da; Shi, Deshi; Yang, Dongfang; Barthel, Benjamin; Koch, Tad H; Yan, Bingfang

    2013-02-01

    Orlistat has been the most used anti-obesity drug and the mechanism of its action is to reduce lipid absorption by inhibiting gastrointestinal lipases. These enzymes, like carboxylesterases (CESs), structurally belong to the α/β hydrolase fold superfamily. Lipases and CESs are functionally related as well. Some CESs (e.g., human CES1) have been shown to hydrolyze lipids. This study was designed to test the hypothesis that orlistat inhibits CESs with higher potency toward CES1 than CES2, a carboxylesterase with little lipase activity. Liver microsomes and recombinant CESs were tested for the inhibition of the hydrolysis of standard substrates and the anticancer prodrugs pentyl carbamate of p-aminobenzyl carbamate of doxazolidine (PPD) and irinotecan. Contrary to the hypothesis, orlistat at 1 nM inhibited CES2 activity by 75% but no inhibition on CES1, placing CES2 one of the most sensitive targets of orlistat. The inhibition varied among some CES2 polymorphic variants. Pretreatment with orlistat reduced the cell killing activity of PPD. Certain mouse but not rat CESs were also highly sensitive. CES2 is responsible for the hydrolysis of many common drugs and abundantly expressed in the gastrointestinal track and liver. Inhibition of this carboxylesterase probably presents a major source for altered therapeutic activity of these medicines if co-administered with orlistat. In addition, orlistat has been linked to various types of organ toxicities, and this study provides an alternative target potentially involved in these toxicological responses.

  7. Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

    Energy Technology Data Exchange (ETDEWEB)

    Crow, J. Allen; Bittles, Victoria; Herring, Katye L.; Borazjani, Abdolsamad [Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762 (United States); Potter, Philip M. [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105 (United States); Ross, Matthew K., E-mail: mross@cvm.msstate.edu [Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762 (United States)

    2012-01-01

    Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC{sub 50} values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k{sub inact}/K{sub I}) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC{sub 50} values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1

  8. cDNA cloning and characterization of the carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L.

    Institute of Scientific and Technical Information of China (English)

    HU Zhen-di; FENG Xia; LIN Qing-sheng; CHEN Huan-yu; LI Zhen-yu; YIN Fei; LIANG Pei; GAO Xi-wu

    2016-01-01

    Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a ful-length cDNA of carboxylesterasepxCCE016b was cloned and exogenously expressed inEscherichia coliat the ifrst time, which contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of thepxCCE016b was signiifcantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore,pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfuly with prokaryotic cel. Western blot analysis showed that it was coded by pxCCE016b. Al the above ifndings provide important information for further functional study, although we are uncertainty

  9. Automated evaluation of pharmaceutically active ionic liquids’ (eco)toxicity through the inhibition of human carboxylesterase and Vibrio fischeri

    Energy Technology Data Exchange (ETDEWEB)

    Costa, Susana P.F.; Justina, Vanessa D. [REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Bica, Katharina; Vasiloiu, Maria [Vienna University of Technology, Institute of Applied and Synthetic Chemistry, A-1060 Vienna (Austria); Pinto, Paula C.A.G., E-mail: ppinto@ff.up.pt [REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Saraiva, M. Lúcia M.F.S., E-mail: lsaraiva@ff.up.pt [REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal)

    2014-01-30

    Highlights: • IL-APIs toxicity on humans and aquatic environment was evaluated by inhibition assays. • The inhibition assays were implemented through automated screening bioassays. • Automation of bioassays enabled a rigorous control of the reaction conditions. • EC{sub 50} obtained provide vital information on IL-APIs safety and potential use as drugs. -- Abstract: The toxicity of 16 pharmaceutical active ionic liquids (IL-APIs) was evaluated by automated approaches based on sequential injection analysis (SIA). The implemented bioassays were centered on the inhibition of human carboxylesterase 2 and Vibrio fischeri, in the presence of the tested compounds. The inhibitory effects were quantified by calculating the inhibitor concentration required to cause 50% of inhibition (EC{sub 50}). The EC{sub 50} values demonstrated that the cetylpyridinium group was one of the most toxic cations and that the imidazolium group was the less toxic. The obtained results provide important information about the safety of the studied IL-APIs and their possible use as pharmaceutical drugs. The developed automated SIA methodologies are robust screening bioassays, and can be used as a generic tools to identify the (eco)toxicity of the structural elements of ILs, contributing to a sustainable development of drugs.

  10. Two homologous carboxylesterase genes from Locusta migratoria with different tissue expression patterns and roles in insecticide detoxification.

    Science.gov (United States)

    Zhang, Jianqin; Ge, Pingting; Li, Daqi; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2015-06-01

    Carboxylesterases (CarEs) play a crucial role in detoxification of xenobiotics and resistance to insecticides in insects. In this study, two cDNAs of CarE genes (LmCesA4 and LmCesA5) were sequenced from the migratory locust, Locusta migratoria. The cDNAs of LmCesA4 and LmCesA5 putatively encoded 538 and 470 amino acid residues, respectively. The deduced amino acid sequences of the two CarE genes showed 45.0% identities, possessed highly conserved catalytic triads (Ser-Glu-His), and clustered in phylogenetic analysis. These results suggest that they are homologous genes. Both CarE genes were expressed throughout the developmental stages. However, LmCesA4 was predominately expressed in the midgut (including the gastric caeca) and fat bodies, whereas LmCesA5 was mainly expressed in the gastric caeca. The in situ hybridization results showed that the transcripts of the two genes were localized in apical and basal regions of the columnar cells in the gastric caeca. Gene silencing followed by insecticide bioassay increased the mortalities of deltamethrin-, malathion-, and carbaryl-treated locusts by 29.5%, 31.0% and 20.4%, respectively, after the locusts were injected with LmCesA4 double-stranded RNA (dsRNA). In contrast, the injection of LmCesA5 dsRNA did not significantly increase the susceptibility of the locusts to any of these insecticides. These results suggest that these genes not only show different tissue expression patterns but also play different roles in insecticide detoxification.

  11. Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus.

    Directory of Open Access Journals (Sweden)

    Linda Grigoraki

    2015-05-01

    Full Text Available The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated.Temephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE, but not target site resistance (altered AChE, with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain; these genes have been previously shown to be involved in temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and temephos resistance, by demonstrating a strong association between survival to temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs, cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance.The identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic

  12. Effects of host plants on insecticide susceptibility and carboxylesterase activity in Bemisia tabaci biotype B and greenhouse whitefly, Trialeurodes vaporariorum.

    Science.gov (United States)

    Liang, Pei; Cui, Jian-Zhou; Yang, Xiu-Qing; Gao, Xi-Wu

    2007-04-01

    Bemisia tabaci (Gennadius) biotype B and the greenhouse whitefly, Trialeurodes vaporariorum (Westwood), have become serious pests of cotton and vegetable crops in China since the early 1990s. In recent years, however, B. tabaci have broken out more frequently and widely than have T. vaporariorum. The B. tabaci biotype B has also developed higher resistance to several insecticides. Here, the effects of four different host plants on the insecticide susceptibility of B. tabaci biotype B and T. vaporariorum have been compared. The LC(50) values of imidacloprid, abamectin, deltamethrin and omethoate in T. vaporariorum reared on cucumber were significantly higher than those in B. tabaci (the LC(50) values in T. vaporariorum were respectively 3.13, 2.63, 2.78 and 6.67 times higher than those in B. tabaci). On the other hand, the B. tabaci population reared on cotton was more tolerant to all four insecticides tested than the T. vaporariorum population from the same host, especially to abamectin (up to 8.4-fold). The effects of the four host plants on the activity of carboxylesterase (CarE) in B. tabaci biotype B and T. vaporariorum were also compared. The results showed that, although the CarE activity of B. tabaci and T. vaporariorum varied depending on the host plants, the B. tabaci population possessed significantly higher CarE activity than the T. vaporariorum population reared on the same host plant. This was especially so on cucumber and cotton, where the CarE activities of the B. tabaci population were over 1.6 times higher than those of T. varporariorum. The frequency profiles for this activity in B. tabaci and T. vaporariorum populations reared on same host plant were apparently different.

  13. Association of a carboxylesterase 1 polymorphism with appetite reduction in children and adolescents with attention-deficit/hyperactivity disorder treated with methylphenidate.

    Science.gov (United States)

    Bruxel, E M; Salatino-Oliveira, A; Genro, J P; Zeni, C P; Polanczyk, G V; Chazan, R; Rohde, L A; Hutz, M H

    2013-10-01

    Carboxylesterase 1 is the enzyme involved in methylphenidate (MPH) metabolism. The aim of this study was to evaluate the association between a -75 T>G polymorphism and appetite reduction in children with attention-deficit/hyperactivity disorder (ADHD). A sample of 213 children with ADHD was investigated. The primary outcome was appetite reduction measured by the Barkley Stimulant Side Effect Rating Scale applied at baseline, at 1 and 3 months of treatment. MPH doses were augmented until no further clinical improvement or significant adverse events occurred. The G allele presented a trend for association with appetite reduction scores (P=0.05). A significant interaction between the G allele and treatment over time for appetite reduction scores was also observed (P=0.03). The G allele carriers presented a higher risk for appetite reduction worsening when compared with T allele homozygotes (odds ratio=3.47, P=0.01). The present results suggest an influence of carboxylesterase 1 -75 T>G polymorphism on the worsening of appetite reduction with MPH treatment in youths with ADHD.

  14. Molecular and functional characterization of cDNAs putatively encoding carboxylesterases from the migratory locust, Locusta migratoria.

    Directory of Open Access Journals (Sweden)

    Jianqin Zhang

    Full Text Available Carboxylesterases (CarEs belong to a superfamily of metabolic enzymes encoded by a number of genes and are widely distributed in microbes, plants and animals including insects. These enzymes play important roles in detoxification of insecticides and other xenobiotics, degradation of pheromones, regulation of neurodevelopment, and control of animal development. In this study, we characterized a total of 39 full-length cDNAs putatively encoding different CarEs from the migratory locust, Locusta migratoria, one of the most severe insect pests in many regions of the world, and evaluated the role of four CarE genes in insecticide detoxification. Our phylogenetic analysis grouped the 39 CarEs into five different clades including 20 CarEs in clade A, 3 in D, 13 in E, 1 in F and 2 in I. Four CarE genes (LmCesA3, LmCesA20, LmCesD1, LmCesE1, representing three different clades (A, D and E, were selected for further analyses. The transcripts of the four genes were detectable in all the developmental stages and tissues examined. LmCesA3 and LmCesE1 were mainly expressed in the fat bodies and Malpighian tubules, whereas LmCesA20 and LmCesD1 were predominately expressed in the muscles and hemolymph, respectively. The injection of double-stranded RNA (dsRNA synthesized from each of the four CarE genes followed by the bioassay with each of four insecticides (chlorpyrifos, malathion, carbaryl and deltamethrin increased the nymphal mortalities by 37.2 and 28.4% in response to malathion after LmCesA20 and LmCesE1 were silenced, respectively. Thus, we proposed that both LmCesA20 and LmCesE1 played an important role in detoxification of malathion in the locust. These results are expected to help researchers reveal the characteristics of diverse CarEs and assess the risk of insecticide resistance conferred by CarEs in the locust and other insect species.

  15. Molecular and functional characterization of cDNAs putatively encoding carboxylesterases from the migratory locust, Locusta migratoria.

    Science.gov (United States)

    Zhang, Jianqin; Li, Daqi; Ge, Pingting; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2014-01-01

    Carboxylesterases (CarEs) belong to a superfamily of metabolic enzymes encoded by a number of genes and are widely distributed in microbes, plants and animals including insects. These enzymes play important roles in detoxification of insecticides and other xenobiotics, degradation of pheromones, regulation of neurodevelopment, and control of animal development. In this study, we characterized a total of 39 full-length cDNAs putatively encoding different CarEs from the migratory locust, Locusta migratoria, one of the most severe insect pests in many regions of the world, and evaluated the role of four CarE genes in insecticide detoxification. Our phylogenetic analysis grouped the 39 CarEs into five different clades including 20 CarEs in clade A, 3 in D, 13 in E, 1 in F and 2 in I. Four CarE genes (LmCesA3, LmCesA20, LmCesD1, LmCesE1), representing three different clades (A, D and E), were selected for further analyses. The transcripts of the four genes were detectable in all the developmental stages and tissues examined. LmCesA3 and LmCesE1 were mainly expressed in the fat bodies and Malpighian tubules, whereas LmCesA20 and LmCesD1 were predominately expressed in the muscles and hemolymph, respectively. The injection of double-stranded RNA (dsRNA) synthesized from each of the four CarE genes followed by the bioassay with each of four insecticides (chlorpyrifos, malathion, carbaryl and deltamethrin) increased the nymphal mortalities by 37.2 and 28.4% in response to malathion after LmCesA20 and LmCesE1 were silenced, respectively. Thus, we proposed that both LmCesA20 and LmCesE1 played an important role in detoxification of malathion in the locust. These results are expected to help researchers reveal the characteristics of diverse CarEs and assess the risk of insecticide resistance conferred by CarEs in the locust and other insect species.

  16. Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor

    Directory of Open Access Journals (Sweden)

    Chan Jeannie

    2008-02-01

    Full Text Available Abstract Background Carboxylesterases (CES perform diverse metabolic roles in mammalian organisms in the detoxification of a broad range of drugs and xenobiotics and may also serve in specific roles in lipid, cholesterol, pheromone and lung surfactant metabolism. Five CES families have been reported in mammals with human CES1 and CES2 the most extensively studied. Here we describe the genetics, expression and phylogeny of CES isozymes in the opossum and report on the sequences and locations of CES1, CES2 and CES6 'like' genes within two gene clusters on chromosome one. We also discuss the likely sequence of gene duplication events generating multiple CES genes during vertebrate evolution. Results We report a cDNA sequence for an opossum CES and present evidence for CES1 and CES2 like genes expressed in opossum liver and intestine and for distinct gene locations of five opossum CES genes,CES1, CES2.1, CES2.2, CES2.3 and CES6, on chromosome 1. Phylogenetic and sequence alignment studies compared the predicted amino acid sequences for opossum CES with those for human, mouse, chicken, frog, salmon and Drosophila CES gene products. Phylogenetic analyses produced congruent phylogenetic trees depicting a rapid early diversification into at least five distinct CES gene family clusters: CES2, CES1, CES7, CES3, and CES6. Molecular divergence estimates based on a Bayesian relaxed clock approach revealed an origin for the five mammalian CES gene families between 328–378 MYA. Conclusion The deduced amino acid sequence for an opossum cDNA was consistent with its identity as a mammalian CES2 gene product (designated CES2.1. Distinct gene locations for opossum CES1 (1: 446,222,550–446,274,850, three CES2 genes (1: 677,773,395–677,927,030 and a CES6 gene (1: 677,585,520–677,730,419 were observed on chromosome 1. Opossum CES1 and multiple CES2 genes were expressed in liver and intestine. Amino acid sequences for opossum CES1 and three CES2 gene products

  17. Toxicity of parathion on embryo and yolk-sac larvae of gilthead seabream (Sparus aurata l.): effects on survival, cholinesterase, and carboxylesterase activity.

    Science.gov (United States)

    Arufe, M Isabel; Arellano, Juana M; Albendín, Gemma; Sarasquete, Carmen

    2010-12-01

    This study was conducted to examine the acute toxicity of the organophosphorus pesticide (OP) parathion on embryos and yolk-sac larvae of gilthead seabream (Sparus aurata), and to investigate the effects of this compound on cholinesterase and carboxylesterase activity of seabream larvae in the phase of endogenous feeding. The 72-h LC50 for yolk-sac larvae (0.523 mg L⁻¹) was about two-fold lower than the 48-h LC50 for embryos (1.005 mg L⁻¹). Parathion significantly inhibited the activity of ChE and CaE activity in yolk sac larvae but there were not significant differences in the sensitivity of both esterases to parathion as inferred by their 72-h IC50 values. Larvae exposed to parathion for 72 h showed a 70% inhibition of the whole body acetylcholinesterase at approximately the LC50.

  18. Understanding Human Carboxylesterase 2

    OpenAIRE

    Lamego, Joana

    2012-01-01

    Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences, Biotechnology The first barrier oral drugs and prodrugs encounter prior to reaching an organism’s systemic circulation is the gastrointestinal (GI) tract, specifically the intestine, which is the primary section for absorption. Therefore, it is fundamental to understand the permeability of the therapeutic agent as well as its potential metabolism by human enterocytes, since biotransformatio...

  19. Molecular cloning, characterization and expression analysis of two juvenile hormone esterase-like carboxylesterase cDNAs in Chinese mitten crab, Eriocheir sinensis.

    Science.gov (United States)

    Xu, Yu; Zhao, Muzi; Deng, Yanfei; Yang, Yuanjie; Li, Xuguang; Lu, Quanping; Ge, Jiachun; Pan, Jianlin; Xu, Zhiqiang

    2017-03-01

    Precise regulation of methyl farnesoate (MF) titer is of prime importance throughout the crustacean life-cycle. Although the synthetic pathway of MF is well-documented, little is known about its degradation and recycling in crustaceans. Juvenile hormone esterase-like (JHE-like) carboxylesterase (CXE) is a key enzyme in MF degradation, thus playing a significant role in regulating the MF titer. We identified and characterized two cDNAs, Es-CXE1 and Es-CXE2, encoding JHE-like CXEs in Chinese mitten crab. Full-length cDNAs of Es-CXE1 and Es-CXE2 encode proteins composed of 584 and 597 amino acids, respectively, both of which contain a typical carboxylesterase domain. Alignment and phylogenetic analyses revealed that the Es-CXEs are highly similar to those of other crustaceans. To further validate their functions, we evaluated the mRNA expression patterns of the Es-CXEs in various tissues and in different physiological conditions. Tissue-specific expression analysis showed that the two Es-CXEs were predominantly expressed in the hepatopancreas and ovaries, which are the major tissues for MF metabolism. Es-CXE2 expression levels in the hepatopancreas and ovaries were about 100 and 25-fold higher, than the respective Es-CXE1 expressions. During ovarian rapid development stage, the global expressions of Es-CXEs were up-regulated in the hepatopancreas and down-regulated in the ovaries. After eyestalk ablation (ESA), the mRNA expressions of the two Es-CXEs were up-regulated in the hepatopancreas, further indicating their potential in degrading MF. Taken together, our results suggest that Es-CXEs, the key component of the juvenile hormone degradation pathway, may play vital roles in the development and reproduction of the Chinese mitten crab.

  20. Comparison of cholin- and carboxylesterase enzyme inhibition and visible effects in the zebra fish embryo bioassay under short-term paraoxon-methyl exposure.

    Science.gov (United States)

    Küster, E; Altenburger, R

    2006-01-01

    The acute zebra fish embryo test (Danio rerio Hamilton-Buchanan, 1822) is an accepted bioassay to assess the toxicity of waste water that may be used for the replacement of testing with adult fish. It is also suggested for chemical hazard characterization and assessment, although only a few groups of substances have yet been studied. Specifically acting substances such as neurotoxic insecticides pose a potentially hazard for non-target fish. To establish whether the proposed zebra fish embryo test protocol and the inhibition of cholinesterases (acetylcholinesterase EC 3.1.1.7, propionylcholinesterase EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) enzymes can be used in a similar fashion for hazard characterization and risk assessment of chemicals and environmental samples, two types of experiments were conducted. Visual effects of exposure to the organophosphate metabolite paraoxon-methyl after 24 and 48 h in the zebra fish embryo test system were analysed with the use of an inverse microscope (rate of mortality, developmental disturbances, heart rate and others). The inhibition to cholinesterases and carboxylesterase was also measured. Enzyme inhibition as a biomarker of exposure was about 70 times more sensitive than the effects in the zebra fish embryo test with an IC50 below 1.2 micromol compared with an EC50 of 91 micromol. The dose-response relationships showed different curve characteristics with a linear increase of enzyme inhibition compared with a sigmoidal curve for the overt effects. Significant overt effects could only be seen at concentrations at which already 80% of the activities of the different esterases were inhibited.

  1. Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus, from India and its possible role in indoxacarb degradation

    Directory of Open Access Journals (Sweden)

    Shanivarsanthe Leelesh Ramya

    2016-06-01

    Full Text Available Abstract Diamondback moth (DBM, Plutella xylostella (Linnaeus, is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13 and adults (n = 12 of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%, followed by bacilli (15.4%. Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%, bacilli (16.7% and flavobacteria (16.7%. Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

  2. Gastrointestinal Degradation of Fumonisin B₁ by Carboxylesterase FumD Prevents Fumonisin Induced Alteration of Sphingolipid Metabolism in Turkey and Swine.

    Science.gov (United States)

    Masching, Sabine; Naehrer, Karin; Schwartz-Zimmermann, Heidi-Elisabeth; Sărăndan, Mihai; Schaumberger, Simone; Dohnal, Ilse; Nagl, Veronika; Schatzmayr, Dian

    2016-03-21

    The mycotoxin fumonisin B₁ (FB₁) is a frequent contaminant of feed and causes various adverse health effects in domestic animals. Hence, effective strategies are needed to prevent the impact of fumonisins on livestock productivity. Here we evaluated the capability of the fumonisin carboxylesterase FumD to degrade FB₁ to its less toxic metabolite hydrolyzed FB₁ (HFB₁) in the gastrointestinal tract of turkeys and pigs. First, an ex vivo pig model was used to examine the activity of FumD under digestive conditions. Within 2 h of incubation with FumD, FB₁ was completely degraded to HFB₁ in the duodenum and jejunum, respectively. To test the efficacy of the commercial application of FumD (FUMzyme) in vivo, female turkeys (n = 5) received either basal feed (CON), fumonisin-contaminated feed (15 mg/kg FB₁+FB₂; FB) or fumonisin-contaminated feed supplemented with FUMzyme (15 U/kg; FB+FUMzyme) for 14 days ad libitum. Addition of FUMzyme resulted in significantly decreased levels of FB₁ in excreta, whereas HFB₁ concentrations were significantly increased. Compared to the FB group (0.24 ± 0.02), the mean serum sphinganine-to-sphingosine (Sa/So) ratio was significantly reduced in the FB+FUMzyme group (0.19 ± 0.02), thus resembling values of the CON group (0.16 ± 0.02). Similarly, exposure of piglets (n = 10) to 2 mg/kg FB₁+FB₂ for 42 days caused significantly elevated serum Sa/So ratios (0.39 ± 0.15) compared to the CON group (0.14 ± 0.01). Supplementation with FUMzyme (60 U/kg) resulted in gastrointestinal degradation of FB₁ and unaffected Sa/So ratios (0.16 ± 0.02). Thus, the carboxylesterase FumD represents an effective strategy to detoxify FB₁ in the digestive tract of turkeys and pigs.

  3. Establishment of Optimum Reaction System of Carboxylesterase from Gypsy Moth (Lymantria dispar)%舞毒蛾羧酸酯酶最佳反应体系的确立1)

    Institute of Scientific and Technical Information of China (English)

    付莉; 胡春祥

    2015-01-01

    In order to study the optimum reaction system of carboxylesterase (CarE) from the gypsy moth (Lymantria dispar), we used orthogonal experiment to measure the effects of the concentrations of carboxylesterase and substrate , pH, reaction temperature and reaction time to the activity of CarE .Through analyzing the data from orthogonal experiment with range analy-sis and analysis of variance , the optimum reaction system of carboxylesterase was ultimately determined as 0.2 beetle· mL-1 of carboxylesterase concentration , 0.2 mmol· L-1 of substrate concentration , 7.5 of pH, 45℃of reaction temperature , and 10 min of reaction time .%为了确定舞毒蛾(Lymantrai dispar)羧酸酯酶(CarE)活性的最佳反应体系,运用正交试验测定了反应体系中酶浓度、底物浓度、pH值、反应温度和反应时间5个因素对CarE活性的影响。极差分析和方差分析显示,舞毒蛾CarE的最优反应体系为酶浓度0.2头· mL-1,底物浓度0.2 mmol· L-1,pH=7.5,反应温度45℃,反应时间10 min。

  4. Identification of amino acids related to catalytic function of Sulfolobus solfataricus P1 carboxylesterase by site-directed mutagenesis and molecular modeling

    Science.gov (United States)

    Choi, Yun-Ho; Lee, Ye-Na; Park, Young-Jun; Yoon, Sung-Jin; Lee, Hee-Bong

    2016-01-01

    The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues. [BMB Reports 2016; 49(6): 349-354] PMID:27222124

  5. Gene Cloning and Characterization of the Geobacillus thermoleovorans CCR11 Carboxylesterase CaesCCR11, a New Member of Family XV.

    Science.gov (United States)

    Espinosa-Luna, Graciela; Sánchez-Otero, María Guadalupe; Quintana-Castro, Rodolfo; Matus-Toledo, Rodrigo Eloir; Oliart-Ros, Rosa María

    2016-01-01

    A gene encoding a carboxylesterase produced by Geobacillus thermoleovoras CCR11 was cloned in the pET-3b cloning vector, sequenced and expressed in Escherichia coli BL21(DE3). Gene sequence analysis revealed an open reading frame of 750 bp that encodes a polypeptide of 250 amino acid residues (27.3 kDa) named CaesCCR11. The enzyme showed its maximum activity at 50 °C and pH 5-8, with preference for C4 substrates, confirming its esterase nature. It displayed good resistance to temperature, pH, and the presence of organic solvents and detergents, that makes this enzyme biotechnologically applicable in the industries such as fine and oleo-chemicals, cosmetics, pharmaceuticals, organic synthesis, biodiesel production, detergents, and food industries. A 3D model of CaesCCR11 was predicted using the Bacillus sp. monoacyl glycerol lipase bMGL H-257 structure as template (PBD code 3RM3, 99 % residue identity with CaesCCR11). Based on its canonical α/β hydrolase fold composed of 7 β-strands and 6 α-helices, the α/β architecture of the cap domain, the GLSTG pentapeptide, and the formation of distinctive salt bridges, we are proposing CaesCCR11 as a new member of family XV of lipolytic enzymes.

  6. Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus.

    Science.gov (United States)

    Grigoraki, Linda; Balabanidou, Vassileia; Meristoudis, Christos; Miridakis, Antonis; Ranson, Hilary; Swevers, Luc; Vontas, John

    2016-07-01

    Temephos is a major organophosphate (OP) larvicide that has been used extensively for the control of Aedes albopictus and Aedes aegypti, the major vectors for viral diseases, such as dengue fever, zika and chikungunya. Resistance to temephos has been recently detected and associated with the upregulation of carboxylesterases (CCEs) through gene amplification, in both species. Here, we expressed the CCEae3a genes which showed the most striking up-regulation in resistant Aedes strains, using the baculovirus system. All CCEae3a variants encoded functional enzymes, with high activity and preference for p-nitrophenyl butyrate, a substrate that was shown capable to differentiate temephos resistant from susceptible Aedes larvae. Enzyme kinetic studies showed that CCEae3as from both Ae. aegypti and Ae. albopictus (CCEae3a_aeg and CCEae3a_alb, respectively) strongly interact with temephos oxon and slowly released the OP molecule, indicating a sequestration resistance mechanism. No difference was detected between resistant and susceptible CCEae3a_aeg variants (CCEae3a_aegR and CCEae3a_aegS, respectively), indicating that previously reported polymorphism is unlikely to play a role in temephos resistance. HPLC/MS showed that CCEae3as were able to metabolize temephos oxon to the temephos monoester [(4-hydroxyphenyl) sulfanyl] phenyl O,O-dimethylphosphorothioate. Western blot and immunolocalization studies, based on a specific antibody raised against the CCEae3a_alb showed that the enzyme is expressed at higher levels in resistant insects, primarily in malpighian tubules (MT) and nerve tissues.

  7. Effect of temperature and sorbitol in improving the solubility of carboxylesterases protein CpCE-1 from Cydia pomonella and biochemical characterization.

    Science.gov (United States)

    Yang, Xueqing; Zhang, Yalin

    2013-12-01

    Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella, CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E. coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni(2+)-NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k cat/K m of 100 s(-1) μM(-1) for α-NA, and the value is 29.78 s(-1) μM(-1) for β-naphthyl acetate. The V max and K m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu(2+) and Mg(2+) significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C. pomonella in vitro. This is the first report of the characterization of CEs protein from C. pomonella.

  8. Effect of two single mutations on malathion degradation by insect carboxylesterases%两个单点突变对昆虫羧酸酯酶降解马拉硫磷的影响

    Institute of Scientific and Technical Information of China (English)

    张柳平; 姚淑敏; 林哲; 崔峰

    2013-01-01

    马拉硫磷是一种高效低毒的有机磷杀虫剂,分子量大且结构特殊,广泛用于农业害虫的防治.羧酸酯酶突变是昆虫对有机磷类杀虫剂产生代谢抗性的重要机制之一.本实验室前期已从棉蚜Aphis gossypii、褐飞虱Nilaparvata lugens、斜纹夜蛾Spodoptera litura、家蚕Bombyx mori、异色瓢虫Harmonia axyridis、赤拟谷盗Tribolium castaneum和西方蜜蜂Apis mellifera中各克隆了一个非特异性羧酸酯酶基因,通过体外定点突变构建了G/A151D和W271L两种突变体,并进行了原核细胞表达和纯化.本实验在体外测定了这7种昆虫野生型和两种突变型羧酸酯酶对马拉硫磷的降解.结果显示:棉蚜、西方蜜蜂、斜纹夜蛾、赤拟谷盗的野生型羧酸酯酶能够降解马拉硫磷,两个突变并不能提高它们的降解活性,而家蚕、异色瓢虫和褐飞虱的野生型羧酸酯酶不能降解马拉硫磷,G/A151D和/或W271L突变能使这些酯酶获得马拉硫磷羧酸酯酶(MCE)的活性,有可能使这些昆虫对马拉硫磷产生抗性.不同物种的MCE活性相差较大,斜纹夜蛾的MCE活性最高,其kcat/Km值为1.8~1.9 L/μmol·min,其次是赤拟谷盗,其Kcat/Km值为0.87 ~0.95 L/μmol·min,其他昆虫的MCE活性相对较低,相差可高达10倍.%Malathion is an efficient but low toxic organophosphate insecticide with a large molecular weight and a special structure.It is widely used in the prevention and control of various agricultural pests.Mutation in carboxylesterases is one of important metabolic resistance mechanisms to organophosphate insecticides in insects.In a previous study,seven non-specific carboxylesterase genes from Aphis gossypii,Nilaparvata lugens,Spodoptera litura,Bombyx mori,Harmonia axyridis,Tribolium castaneum and Apis mellifera,respectively,were cloned,mutated at position 151 or 271 and expressed in Escherichia coli.In this experiment,the hydrolysis of the purified recombinant proteins of the seven

  9. Oral delivery mediated RNA interference of a carboxylesterase gene results in reduced resistance to organophosphorus insecticides in the cotton Aphid, Aphis gossypii Glover.

    Directory of Open Access Journals (Sweden)

    You-Hui Gong

    Full Text Available BACKGROUND: RNA interference (RNAi is an effective tool to examine the function of individual genes. Carboxylesterases (CarE, EC 3.1.1.1 are known to play significant roles in the metabolism of xenobiotic compounds in many insect species. Previous studies in our laboratory found that CarE expression was up-regulated in Aphis gossypii (Glover (Hemiptera: Aphididae adults of both omethoate and malathion resistant strains, indicating the potential involvement of CarE in organophosphorus (OP insecticide resistance. Functional analysis (RNAi is therefore warranted to investigate the role of CarE in A. gossypii to OPs resistance. RESULT: CarE expression in omethoate resistant individuals of Aphis gossypii was dramatically suppressed following ingestion of dsRNA-CarE. The highest knockdown efficiency (33% was observed at 72 h after feeding when dsRNA-CarE concentration was 100 ng/µL. The CarE activities from the CarE knockdown aphids were consistent with the correspondingly significant reduction in CarE expression. The CarE activity in the individuals of control aphids was concentrated in the range of 650-900 mOD/per/min, while in the individuals of dsRNA-CarE-fed aphids, the CarE activity was concentrated in the range of 500-800 mOD/per/min. In vitro inhibition experiments also demonstrated that total CarE activity in the CarE knockdown aphids decreased significantly as compared to control aphids. Bioassay results of aphids fed dsRNA-CarE indicated that suppression of CarE expression increased susceptibility to omethoate in individuals of the resistant aphid strains. CONCLUSION: The results of this study not only suggest that ingestion of dsRNA through artificial diet could be exploited for functional genomic studies in cotton aphids, but also indicate that CarE can be considered as a major target of organophosphorus insecticide (OPs resistance in A. gossypii. Further, our results suggest that the CarE would be a propitious target for OPs resistant

  10. Identification and expression analysis of carboxylesterase gene BrnCarE.9 in Bombyx mori%家蚕羧酸酯酶基因BmCarE-9的鉴定与表达分析

    Institute of Scientific and Technical Information of China (English)

    林超; 李兵; 王东; 赵国栋; 卫正国; 陈玉华; 沈卫德

    2011-01-01

    Carboxylesterase is a multifunctional superfamily. To determine the function of carboxylesterase in different tissues of Bombyx mori, we cloned a carboxylesterase gene named BmCarE-9 (GenBank accession no. EU523534) from B. mori by using rapid amplification of cDNA ends (RACE) and reverse transcription polymerase chain reaction (RT-PCR) methods. BmCarE-9 contains a 1 680 bp open reading frame (ORF), which encodes 559 amino acids. This cDNA-deduced protein BmCarE-9 has a predicted molecular weight (MW) of 64.2 kD, and isoelectric point (pi) of 7.13. Structure analysis showed that BmCarE-9 has a similar catalytic triad in which two residues have changed. We investigated the developmental expression patterns of BmCarE-9 in different tissues of the Day-3 5th instar larvae and in silk glands of different day-old 5th instar larvae by real-time quantitative PCR. The results showed that BmCarE9 was expressed specifically in silk glands at high levels, and mainly expressed in the median and posterior silk glands. Furthermore, the expression of BmCarE-9 increased as silk glands developed, and decreased at the end of 5th instar stage. It is so inferred that BmCarE-9 might be involved in the development of silk glands or the synthesis of silk proteins.%羧酸酯酶是一个多功能超家族酶类,为研究羧酸酯酶基因在家蚕Bombyx mori组织中的功能,利用5′/3′RACE和RT-PCR方法克隆了一个家蚕羧酸酯酶基因BmCarE-9,其GenBank登录号为EU523534.该基因含有一个1 680 bp的ORF,编码559个氨基酸.BmCarE-9预测的分子量64.2kD,等电点7.13,结构分析表明BmCarE-9存在一个类似的催化三联体,其中两个残基发生改变.利用实时荧光定量PCR方法研究了该基因在家蚕5龄第3天幼虫不同组织以及在5龄期各日龄幼虫丝腺组织中的表达水平.结果表明,该基因在丝腺中特异性高表达,且主要在中部和后部丝腺中表达.该基因在5龄期随着丝腺的生长发育表达量逐渐

  11. Mutation of residues 423 (Met/Ile), 444 (Thr/Met), and 506 (Asn/Ser) confer cholesteryl esterase activity on rat lung carboxylesterase. Ser-506 is required for activation by cAMP-dependent protein kinase.

    Science.gov (United States)

    Wallace, T J; Kodsi, E M; Langston, T B; Gergis, M R; Grogan, W M

    2001-08-31

    Site-directed mutagenesis is used to identify amino acid residues that dictate reported differences in substrate specificity between rat hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) and rat lung carboxylesterase (LCE), proteins differing by only 4 residues in their primary sequences. Beginning with LCE, the substitution Met(423) --> Ile(423) alone or in combination with other mutations increased activity with p-nitrophenylcaprylate (PNPC) relative to more hydrophilic p-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr(444) --> Met(444) was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn(506) --> Ser(506), creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase, a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase activity by the mutated LCE. The substitution Gln(186) --> Arg(186) selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that provide insight into the effects of these mutations on substrate specificity.

  12. Application of carboxylesterase in beta-cyfluthrin resistance surveillance of Musca domestica%羧酸酯酶用于家蝇高效氟氯氰菊酯抗性监测的研究

    Institute of Scientific and Technical Information of China (English)

    陶卉英; 马红梅; 柳小青; 李卫民; 郭学俭

    2011-01-01

    目的 探讨羧酸酯酶(CarE)与家蝇高效氟氯氰菊酯抗性之间的关系,并将CarE应用于不同生境家蝇高效氟氯氰菊酯抗性的早期检测.方法 采用微量点滴法进行生物测定;采用Asperen方法进行CarE活性测定.结果 生物测定结果表明,除垃圾中转站家蝇种群外,居民区、餐饮店和农贸市场家蝇种群对高效氟氯氰菊酯产生了不同程度的耐药性或抗药性,抗性指数依次为1.84、15.31、3.19、8.84倍.CarE活性测定结果表明,除垃圾中转站种群外,其它3个家蝇野外种群与敏感品系相比显著升高(P<0.05),而且家蝇CarE的比活力与其对高效氟氯氰菊酯的抗性之间呈显著的正相关.对不同生境家蝇种群CarE个体频率分布情况的研究也发现,农贸市场、餐饮店和居民区家蝇种群与敏感品系间有明显的重叠现象.此外,3个家蝇种群高活性的个体频率分别为居民区种群75%,农贸市场种群72%,餐饮店种群54%.结论 CarE活性的升高与家蝇高效氟氯氰菊酯的抗性有关;不同个体CarE活性分布情况表明农贸市场、餐饮店和居民区家蝇种群基本上是抗性杂合种群,与生物测定结果基本相符.此外,根据不同生境家蝇种群中高活性个体频率的发展趋势,建议居民区在家蝇防制时应限制使用高效氟氯氰菊酯,换用或轮用其他杀虫剂,以避免或延缓抗性的发展;其他生境应慎用高效氟氯氰菊酯.%Objective To determine the association between carboxylesterase (CarE) and beta - cyfluthrin resistance in Musca domestica in various habitats, and monitor this resistance at an early phase by using carboxylesterase. Methods Biological assessment was conducted using dripping titration and the activity of carboxylesterase was determined by the Asperen method. Results Except for the population in transfer stations, the three populations of M. Domestica in residential areas, restaurants and fanner's market presented

  13. 华北大黑鳃金龟Holotrichia oblita中肠羧酸酯酶基因HoCL1的克隆与序列分析%Molecular cloning and sequence analysis of HoCL1 carboxylesterase cDNAs from the midgut of Holotrichia oblita

    Institute of Scientific and Technical Information of China (English)

    高勇; 周洪旭; 郑桂玲; 李长友

    2012-01-01

    The peritrophic membrane protein polyclonal antiserum from Trichoplusia ni was used to screen cDNA expression library of the migut of Holotrichia oblita, and a cDNA clone encoding carboxylesterase named HoCLl was obtained. The sequence analysis indicated that the opening reading frame of HoCLl is 1599 bp in length encoding 532 amino acid residues of the proteinase with predicted molecular weight 59. 5 kDa and p/ 4. 5. HoCLl has conserved domains of carboxylesterases with a disulfide bond formation site,a serine activity center,only three cysteine residues and catalytic activity centers of Ser207, Asp333 and His422, and with no nitrogen linked glycosylation sites and oxygen glycosylation sites. Based on the amino acid sequence homology analysis and conserved domain analysis, HoCLl belongs to B esterase and is mostly similar to the carboxylesterase of Tribolium castaneum,but the similarity is only 35. 2%. In the sequence alignments and phylogenetic tree with other insect carboxylesterases, the sequence conservation of COOH-terminal amino acid in HoCLl is low,but the amino acid sequence near the N terminus is highly conserved. HoCLl clusters with the carboxylesterases of T. castaneum and Harmonia axyridis. Cloning and identification of HoCLl laid the foundation for the study of the expression and function in vivo.%利用粉纹夜蛾(Trichoplusia ni)围食膜蛋白多克隆抗体,从已构建的华北大黑鳃金龟Holotrichia oblita中肠cDNA表达文库中筛选得到1个编码羧酸酯酶的cDNA克隆HoCL1,其开放阅读框(ORF)长1 599 bp,编码532个氨基酸,推导的蛋白质分子质量为59.5 kDa,等电点(pI)为4.5.HoCL1蛋白具有羧酸酯酶的保守结构域:1个二硫键形成的位点和1个丝氨酸活性中心,三联体催化活性中心位于Ser207、Asp333和His422上,不合有氮联糖基化位点和氧联糖基化位点,只含有3个半胱氨酸残基.依据氨基酸序列同源性分析和保守结构域分析,HoCL1属

  14. 棉铃虫酯酶突变体的构建、表达及酶促动力学特性%Construction, expression and activities of mutant carboxylesterases from Helicoverpa armigera

    Institute of Scientific and Technical Information of China (English)

    李永强; 吴美玲; 马志卿; 冯俊涛; 张兴

    2014-01-01

    The cotton bollworm , Helicoverpa armigera ( Hübner),is a major pest of many agricultural crops around the world , and many of the classes of chemical insecticides are widely used for its control such as organophosphates ( OPs) , synthetic pyrethroids ( SPs) , and so on . Currently H .armigera has developed serious resistance to OPs and SPs all over the world . Carboxylesterases ( CarEs) are a multi-gene family of enzymes that hydrolyze a diverse range of carboxylesters and are frequently implicated in the resistance of insects . Gene mutations of CarEs are a major mechanism of insects for the development of resistance to OPs in the OP-resistant Diptera pests like Musca domestica and Lucilia cuprina . It involves the substitution of a single amino acid within the active site of the esterase which convert it to an OP hydrolyase . However , no resembled mutation in nature was reported in the Lepidopterapestslikecottonbollworm,H.armigeratodate.Hence,followingourpreviousstudiestheaimofthe current work was to elucidate the effects of specific point mutations of the carboxylesterases from H . armigera on the kinetic properties of the enzymes . Two CarEs , 001F and 001G from H . armigera , were induced to mutate at positions 127 ( A → D) or 238 (F → L) using a site-directed mutagenesis technique . They were then expressed with the baculovirus expression vector system (BEVS) . The kinetic assays with α-naphthyl acetate ( α-NA) and para-nitrophenyl acetate ( p-NA) were carried out for all mutants using a spectrophotometer . The results showed that the A127D mutations had dramatically reduced the hydrolytic activities of the CarEs toward the two substrates . The mutants all showed lower affinities to the substrates as the Km values were at least 1.6-fold higher than those of the wild type enzymes , and the kcat values (6.5 52.1 s- 1 ) were decreased obviously , which were between 4- and 20-fold lower than those of the wild type enzymes . What’s more , the rate

  15. Identification and expression profiles of Cnaphalocrocis medinalis carboxylesterase genes%稻纵卷叶螟羧酸酯酶基因的鉴定与表达谱分析

    Institute of Scientific and Technical Information of China (English)

    刘苏; 冯明峰; 何梦竹

    2015-01-01

    Objectives] To identify carboxylesterase genes (CarEs) in Cnaphalocrocis medinalis and determine their expression profiles in various adult tissues. [Methods] C. medinalis transcriptome datasets were searched to identify CarEs. Bioinformatic software was used to analyze the identified CarE sequences, and a real-time quantitative PCR assay was performed to investigate their relative expression levels. [Results] A total of 15 CarEs were identified from the C. medinalis transcriptome datasets and named CmCarE1-CmCarE15. Of these, CmCarE12 lacked the 3¢-region whereas the other 14 sequences contained complete open reading frames (ORFs). With the exception of CmCarE11, protein encoded by these CmCarE genes showed features typical of CarEs, including a conserved pentapeptide, catalytic triad, and oxyanion hole. Phylogenetic analysis showed that the 15 CmCarEs fell into different clades;CmCarE13, CmCarE14 and CmCarE15 fell into the“intracellular catalytic class”, whereas the other 12 CmCarEs fell into the“secreted catalytic class”. CmCarE1, CmCarE2, CmCarE3, CmCarE7, CmCarE8, CmCarE10 and CmCarE13 were specifically expressed in the adult abdomen, whereas CmCarE9 was specifically expressed in male and female antennae. Other genes were not tissue-specific. [Conclusion] CmCarE1, CmCarE2, CmCarE3, CmCarE7, CmCarE8, CmCarE10 and CmCarE13 might possibly be involved in the metabolism of endobiotic and xenobiotic compounds, whereas CmCarE9 might be involved in the degradation of odorant molecules.%【目的】鉴定稻纵卷叶螟Cnaphalocrocis medinalis的羧酸酯酶基因,并检测这些基因在成虫不同组织中的表达模式。【方法】从稻纵卷叶螟转录组中搜索羧酸酯酶基因,使用生物信息学软件对所获得的基因序列进行分析,使用荧光定量PCR检测这些基因在各组织中的相对表达水平。【结果】获得了15个羧酸酯酶基因,分别命名为CmCarE1~CmCarE15。其中CmCarE12缺少3′区域,其余14

  16. Expression of Two Carboxylesterase Genes (001f and 001g) from Helicoverpa armigera Using Baculovirus Expression Vector System(BEVS)and Analysis of Their Enzymatic Activity%两个棉铃虫酯酶基因(001f和001g)在杆状病毒表达载体系统(BEVS)中的表达及酶活分析

    Institute of Scientific and Technical Information of China (English)

    李永强; 袁国瑞; 张兴

    2012-01-01

    Metabolic detoxification of carboxylesterases (CarEs) is a major mechanism for the development of resistance in insects to organophosphates (Ops), synthetic pyrethroids (SPs) and carbamates (CBs). In order to elucidate the role of CarE genes from Helicoverpa armigera in metabolic resistance, two CarE genes, Oolf and 001g,were successfully expressed in the Sf9 insect cell lines using the Baculovirus Expression Vector System (BEVS). Both of the expressed CarEs were subjected to native PAGE and stained with artificial substrate a-naphthyl acetate (a-NA), and showed high activity against cx-NA according to the darker stained bands. The resulting stained bands (closely together) indicated that the relative mobility (Rm) of CarEs fell in two zones (0.27 ~0.36 and 0.37 -0.48). The kinetic assays with a-NA and para-nitrophyl acetate (p-NA) using a spectrophotometer showed the two CarEs had high affinity to the former substrate (Xm=(3.9±0.6) and (3.3±0.4) |xmol/L, respectively). They also produced a considerably high rate constant (kcat/Km= (26.5+1.9) and (95±9.3) |xmol"1*s"1*L, respectively) agianst a-NA, but a relatively lower rate constant against p-NA (&cai/X77i=(4.47±0.33) and (12.8±0.10) (imol"1*s"1*L, respectively) compared with that of a-NA. It's showed that the two CarEs had high hydrolytic activity against the artificial substrates. Thus, 00IF and 001G esterases are probabaly associated with metabolic resistance of insecticides, and may have relatively high detoxification activity against some traditional chemical insecticdes such as Ops, SPs and CBs.%昆虫羧酸酯酶(carboxylesterases,CarEs)对有机磷、拟除虫菊酯、氨基甲酸酯等三大类化学杀虫剂的代谢解毒作用是昆虫代谢抗性产生的一种重要机制.为明确棉铃虫(Helicoverpa armigera)两个酯酶基因001f和001g在代谢抗性中的作用,本研究基于杆状病毒表达载体系统(BEVS)在草地夜蛾(Spodoptera frugiperda)Sf9细胞系

  17. 寄主植物对草地螟幼虫的营养利用和中肠酯酶活性的影响%Effects of Host Plants on Nutritional Utilization and the Carboxylesterase Activities in the Larvae of Loxostege sticticalis L

    Institute of Scientific and Technical Information of China (English)

    范锦胜; 张李香; 王贵强; 顾鑫; 王明月

    2011-01-01

    In order to illustrate the effects of different plant hosts on nutritional utilization and activity of the carboxylesterase of the larvae of Loxostege sticticalis L., L. Sticticalis L. Was fed respectively with Chenopodium album L., Glycine max L., Medicago sativa L., Beta vulgaris L., Helianthus animus L.. The results showed that there were significant differences in the nutrient status and activity of the carboxylesterase of the insect fed with 5 different plants. The relative growth rate (RGR=0.91) and the approximate digestibility (AZ)=63.89%) of 3th instar larvae fed with the lambsquarter were significantly higher than those with the soybean, alfalfa, sugarbeet and sunflower. The efficiency of conversion of ingested food (EC/=24.65%, 24.96%) and the efficiency of conversion of digested food (ECD=3%.70%, 41.41%) had no significant differences in larvae fed with the lambsquarter and soybean. The efficiency of conversion of ingested food (EC/=10.63%) and the efficiency of conversion of digested food (ECD=25.89%) of the larvae fed with alfalfa were significantly lower than those fed with the other 4 plants. Preliminary physiological reaction of testing 5* instar larvae showed that the contents of protein in haemolymph of the larvae fed with the soybean were significantly higher than those fed with the other four plants and the contents of protein in mid-gut of the larvae fed with the sunflower were the highest. The esterase in mid-gut of the larvae fed with the lambsquarter and soybean were\\significantly higher than those fed with the other 3 plants. The results suggested that lambsquarter was considered the most appropriate host, followed by soybean, sugarbeet, sunflower and alfalfa.%分别用藜(Chenopodium album L.)、大豆(Glycine max L.)、苜蓿(Medicago sativa L.)、甜菜(Beta vulgans L.)和向日葵(Helianthus annuus L.)饲养草地螟,测定不同食料植物对草地螟幼虫的营养利用及中肠酯酶活性的影响.结果表明:取食灰菜的3

  18. 氯氰菊酯和残杀威亚致死剂量对致倦库蚊羧酸酯酶活性的影响%Effects of cypermethrin and propoxur at sublethal doses on the carboxylesterase activity in Culex pipiens quinqusfasciatus

    Institute of Scientific and Technical Information of China (English)

    柳小青; 刘仰青; 熊志伟; 马红梅

    2011-01-01

    Objective To determine the changes of carboxylesterase (CarE) activity in Cidex pipiens quinqusfasciatus treated with cypermethrin and propoxur at sublethal doses. Methods The enzyme activities were measured in vitro with an ultraviolet-visible spectrophotometer. Results After 24 h treatment, the enzymatic activity of resistant strains was 1.19 times that of sensitive strains. Significant inhibition was noted in all treatment groups after 48 h. Compared with the 24 h treatment group, the enzymatic activity of the control group, propoxur at LC20, propoxur at LC40, cypermethrin at LC20, and cypermethrin at LC40 decreased by 24.71%, 38.42%, 97.42%, 90.77% and 95.76%, respectively. The decreases in all treated groups were more significant than in the control group. Activities in resistant strains treated with cypermethrin and propoxur at sublethal doses for 48 h were significantly different from the control group, except for the group treated with LC20 propoxur. Compared with the control group, the specific activity was 11.6254 nmol/(mgpro · min) in the group treated with LC40 of propoxur, indicating significant inhibitory effect. In the groups treated with LC20 and LC40 of cypermethrin, the activity was 55.8868 and 54.5530 nmol/ (mgpro · min), respectively, suggesting significant inductive effects. No difference was noted between the two 24 h and 48 h treatment groups. Compared with the 24 h treatment group, the specific activity of the control group, propoxur (at LC20 and LC40) and cypermethrin (at LC40) treated groups decreased by 60.71%, 59.14%, 23.68% and 47.87%, respectively, whereas the cypermethrin LC20 group increased by 23.89%. The decrease in the control group was lower than those in the treatment groups. Conclusion Different levels of carboxylesterase activity were observed in sensitive and resistant Cx. Pipiens quinqusfasciatus when treated with sublethal doses of cypermethrin and propoxur. Thus, indicating their different inhibitory effects and that a

  19. Genome sequence of carboxylesterase, carboxylase and xylose isomerase producing alkaliphilic haloarchaeon Haloterrigena turkmenica WANU15

    Directory of Open Access Journals (Sweden)

    Samy Selim

    2016-03-01

    Full Text Available We report draft genome sequence of Haloterrigena turkmenica strain WANU15, isolated from Soda Lake. The draft genome size is 2,950,899 bp with a G + C content of 64% and contains 49 RNA sequence. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LKCV00000000.

  20. Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases.

    Science.gov (United States)

    Hajighasemi, Mahbod; Nocek, Boguslaw P; Tchigvintsev, Anatoli; Brown, Greg; Flick, Robert; Xu, Xiaohui; Cui, Hong; Hai, Tran; Joachimiak, Andrzej; Golyshin, Peter N; Savchenko, Alexei; Edwards, Elizabeth A; Yakunin, Alexander F

    2016-06-13

    Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.

  1. MOLECULAR MODELS OF PARATHYROID METABOLISM BY CARBOXYLESTERASES: DIFFERENTIAL EFFECTS DUE TO STEREOCHEMISTRY.

    Science.gov (United States)

    PARATHYROIDS ARE A CHEMICAL CLASS OF WIDELY USED INSECTICIDES, & AT LEAST 16 CHEMICALS IN THIS CLASS ARE REGISTERED FOR USE IN THE US. IN ORDER TO EXTRAPOLATE THE KNOWLEDGE FROM RODENTS TO HUMANS, PHYSIOLOGICALLY-BASED PHARMACOKINETIC (pbpk) MODELS ARE CURRENTLY BEING DEVELOPED.

  2. Characterization of an antennal carboxylesterase from the pest moth Spodoptera littoralis degrading a host plant odorant.

    Directory of Open Access Journals (Sweden)

    Nicolas Durand

    Full Text Available BACKGROUND: Carboxyl/cholinesterases (CCEs are highly diversified in insects. These enzymes have a broad range of proposed functions, in neuro/developmental processes, dietary detoxification, insecticide resistance or hormone/pheromone degradation. As few functional data are available on purified or recombinant CCEs, the physiological role of most of these enzymes is unknown. Concerning their role in olfaction, only two CCEs able to metabolize sex pheromones have been functionally characterized in insects. These enzymes are only expressed in the male antennae, and secreted into the lumen of the pheromone-sensitive sensilla. CCEs able to hydrolyze other odorants than sex pheromones, such as plant volatiles, have not been identified. METHODOLOGY: In Spodoptera littoralis, a major crop pest, a diversity of antennal CCEs has been previously identified. We have employed here a combination of molecular biology, biochemistry and electrophysiology approaches to functionally characterize an intracellular CCE, SlCXE10, whose predominant expression in the olfactory sensilla suggested a role in olfaction. A recombinant protein was produced using the baculovirus system and we tested its catabolic properties towards a plant volatile and the sex pheromone components. CONCLUSION: We showed that SlCXE10 could efficiently hydrolyze a green leaf volatile and to a lesser extent the sex pheromone components. The transcript level in male antennae was also strongly induced by exposure to this plant odorant. In antennae, SlCXE10 expression was associated with sensilla responding to the sex pheromones and to plant odours. These results suggest that a CCE-based intracellular metabolism of odorants could occur in insect antennae, in addition to the extracellular metabolism occurring within the sensillar lumen. This is the first functional characterization of an Odorant-Degrading Enzyme active towards a host plant volatile.

  3. Population Pharmacokinetics of Methylphenidate in Healthy Adults Emphasizing Novel and Known Effects of Several Carboxylesterase 1 (CES1) Variants

    DEFF Research Database (Denmark)

    Lyauk, Y K; Stage, C; Bergmann, T K;

    2016-01-01

    The aim of this study was to identify demographic and genetic factors that significantly affect methylphenidate (MPH) pharmacokinetics (PK), and may help explain interindividual variability and further increase the safety of MPH. d-MPH plasma concentrations, demographic covariates, and carboxyles......The aim of this study was to identify demographic and genetic factors that significantly affect methylphenidate (MPH) pharmacokinetics (PK), and may help explain interindividual variability and further increase the safety of MPH. d-MPH plasma concentrations, demographic covariates...

  4. Prognostic impact of carboxylesterase 1 gene variants in patients with congestive heart failure treated with angiotensin-converting enzyme inhibitors

    DEFF Research Database (Denmark)

    Nelveg-Kristensen, Karl E; B. Madsen, Majbritt; Torp-Pedersen, Christian;

    2016-01-01

    with congestive heart failure (CHF). METHODS: Danish patients with chronic CHF enrolled in the previously reported Echocardiography and Heart Outcome Study were categorized according to their CES1 variants and followed up for up to 10 years. Risk for cardiovascular death and all-cause death was modeled by Cox...

  5. Liver-specific expression of carboxylesterase 1g/esterase-x reduces hepatic steatosis, counteracts dyslipidemia and improves insulin signaling.

    Science.gov (United States)

    Bahitham, Wesam; Watts, Russell; Nelson, Randal; Lian, Jihong; Lehner, Richard

    2016-05-01

    Ces1g/Es-x deficiency in mice results in weight gain, insulin resistance, fatty liver and hyperlipidemia through upregulation of de novo lipogenesis and oversecretion of triacylglycerol (TG)-rich lipoproteins. Here, we show that restoration of Ces1g/Es-x expression only in the liver significantly reduced hepatic TG concentration accompanied by decreased size of lipid droplets, reduced secretion of very low-density lipoproteins and improved insulin-mediated signal transduction in the liver. Collectively, these results demonstrate that hepatic Ces1g/Es-x plays a critical role in limiting hepatic steatosis, very low-density lipoprotein assembly and in augmenting insulin sensitivity.

  6. Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents

    DEFF Research Database (Denmark)

    Shi, Yuping; Pan, Yingjie; Li, Bailin;

    2013-01-01

    ABSTRACT: BACKGROUND: BioH is one of the key enzymes to produce the precursor pimeloyl-ACP to initiate biotin biosynthesis de novo in bacteria. To date, very few bioH genes have been characterized. In this study, we cloned and identified a novel bioH gene, bioHx, from an environmental metagenome ...

  7. Rhipicephalus (Boophilus) microplus strain Deutsch, 5 BAC clone sequencing, including two encoding Cytochrome P450s and one encoding CzEst9 carboxylesterase

    Science.gov (United States)

    The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and difficult to assemble and analyze. BAC clones give insight into the genome struct...

  8. 嗜热脂肪芽孢杆菌羧酸酯酶的异源表达及酶学性质研究%Heterologous Expression and Characterization of The Carboxylesterase From Geobacillus stearothermophilus

    Institute of Scientific and Technical Information of China (English)

    孙锦霞; 刘钟滨

    2010-01-01

    运用生物信息学技术从嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)CICC 20156中克隆获得羧酸酯酶基因,构建黑曲霉和毕氏酵母表达质粒,将重组质粒分别转化毕氏酵母GS115和黑曲霉pyrG基因缺陷株M54.SDS-PAGE和Westernblot检测显示:携带His标记的外源蛋白在转化真茼宿主中均获得了高效分泌性表达,毕氏酵母和黑曲霉表达的外源蛋白分子质量均约为29ku,蛋白质浓度分别为30.7mg/L和15.3mg/L.生物学活性测定表明,毕氏酵母与黑曲霉表达的羧酸酯酶单位蛋白酶活分别为22 671 U/mg和21 438 U/mg.酶学性质研究显示,两种表达系统表达的重组羧酸酯酶的酶学特性基本一致,它们在40~70℃范围内均显示较好的酶活性,最适反应温度为60℃.70℃处理30min,毕氏酵母和黑曲霉表达重组羧酸酯酶残余酶活分别为76.7%和67.6%,显示出良好的热稳定性.在pH 6.5~8.5的范围内显示较高酶活性,最适pH为8.0.上述研究首次实现了具有良好热稳定性的嗜热脂肪芽孢杆菌羧酸酯酶在黑曲霉和毕氏酵母中高效异源分泌性表达,其中毕氏酵母羧酸酯酶的产量要高于黑曲霉的酶产量,但考虑到重组黑曲霉表达外源性蛋白无需使用任何诱导剂,黑曲霉菌表达热稳定性羧酸酯酶可能具有更好的应用前景.

  9. 易错PCR法提高土芽孢杆菌ZH1羧酸酯酶的热稳定性%Improving thermal stability of Geobacillus sp.ZH1 carboxylesterase by error-prone PCR

    Institute of Scientific and Technical Information of China (English)

    刘韩; 吴丽云; 高贺; 倪辉; 蔡慧农; 朱艳冰

    2015-01-01

    [目的]对土芽孢杆菌(Geobacillus sp.)ZH1的羧酸酯酶基因进行定向进化,筛选得到酶热稳定性提高的突变酶.[方法]利用易错PCR技术向羧酸酯酶基因中随机引入突变,建立酶基因突变文库,筛选获得热稳定性提高的突变体,并对突变酶进行诱导表达、纯化及部分酶学性质研究.[结果]通过筛选,获得羧酸酯酶热稳定性提高的突变菌株65.序列分析表明,突变酯酶65有2个氨基酸发生了改变,包括T113S和M160K.突变酶的三维结构模拟显示,突变T113S位于酶分子的第5个β-折叠上;突变M160K处在酶分子第5个和第6个α-螺旋之间的环结构上,位于酶分子表面,突变后的Lys160与邻近的Thr162形成一个额外氢键.在90℃下,突变酶65和亲本酶的半衰期分别为3.1h和1.9h,表明筛选到的突变酶65比亲本酶的热稳定性好.[结论]基于易错PCR技术对Geobacillus sp.ZH1羧酸酯酶的热稳定性进行了定向进化,对改善酶的性质、扩大酯酶的应用范围,以及研究酯酶的结构与功能的关系具有重要意义.

  10. 一株耐热菌的产酶研究及其羧酸酯酶基因的克隆%Enzyme Production of a Thermophilic Strain and Cloning of Its Carboxylesterase Gene

    Institute of Scientific and Technical Information of China (English)

    朱艳冰; 王国红; 陈少娟; 刘光明

    2011-01-01

    将一株近海温泉耐热菌ZH1的16S rDNA克隆至pUCm-T载体进行测序,测序结果在NCBI 上进行BLAST分析,并构建了系统发育树,将该菌株归属为Geobacillus sp.ZH1.在65℃条件下的产酶定性实验结果表明,该菌株产脂肪酶、木聚糖酶和碱性磷酸酶.以菌株ZH1的基因组DNA为模板,使用羧酸酯酶简并引物进行PCR扩增,将目的基因克隆至pUCm-T载体后进行测序,得到了741bp的DNA片段.BLAST分析显示,目的基因预测编码羧酸酯酶,包含246个氨基酸.

  11. Gene : CBRC-MMUS-11-0132 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available ERASE) (MONOBUTYRASE) (COCAINE ESTERASE) (PROCAINE ESTERASE) (METHYLBUTYRASE) ......d library, clone:A430088E12 product:similar to CARBOXYLESTERASE PRECURSOR (EC 3.1.1.1) (ALI-ESTERASE) (B-EST

  12. Comparison of carboxylesterase acitivity between the resistant and susceptible strains of Tribolium castaneum to phosphine%赤拟谷盗的磷化氢敏感和抗性品系体内羧酸酯酶的活性比较

    Institute of Scientific and Technical Information of China (English)

    王殿轩; 原锴; 高希武

    2010-01-01

    本文比较测定了赤拟谷盗Tribolium castaneum(Herbst)的磷化氢抗性(Rf=327)和敏感品系害虫的羧酸酯酶活性,研究了该害虫同一品系不同个体间羧酸酯酶的活性差异,比较了两品系害虫在系列磷化氢浓度下熏蒸24 h和6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的羧酸酯酶活性.主要结果为:未熏蒸的抗性害虫幼虫和蛹体内的羧酸酯酶活性分别高于敏感品系的1.37和1.16倍;敏感和抗性害虫同品系内不同个体间羧酸酯酶活性分布频率都存在明显差异,抗性害虫中酶活性大的个体数量占的比例较大;磷化氢浓度分别为0.69×10-2、2.78×10-2、5.56×10-2、8.33×10-2和11.11×10-2mg/L时都可导致敏感害虫羧酸酯酶的活性降低,但活性受抑制的程度不因浓度高低呈相应的增减.浓度分别为5.56×10-2、11.1l×10-2、13.89×10-2、20.83×10-2和27.78×10-2mg/L的熏蒸中抗性害虫体内酶活性增加,且活性增高的程度与浓度增减也不呈对应变化.在6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的结果中,敏感害虫的酶活性随时间延长而下降,抗性害虫的活性则随时间延长而增大.研究表明赤拟谷盗对磷化氢的抗性可能与羧酸酯酶的活性增加有关.

  13. Resistance to permethrin and activity of carboxylesterase in Aedes albopictus in Pingshan New District, Shenzhen City%深圳市坪山新区白纹伊蚊对氯菊酯的抗性及羧酸酯酶活性研究

    Institute of Scientific and Technical Information of China (English)

    吴能简; 陈伟文; 吴崧霖; 罗娘胡

    2016-01-01

    目的 了解深圳市坪山新区白纹伊蚊野外种群对氯菊酯抗性及羧酸酯酶活性水平,为科学合理使用杀虫剂提供数据支持.方法 采用幼虫浸渍法对白纹伊蚊进行氯菊酯抗药性测定,同时采用酶联免疫分析法对野外及敏感品系白纹伊蚊进行羧酸酯酶活性测试,分析比较野外品系和敏感品系羧酸酯酶活性的差异.结果 坪山新区白纹伊蚊对氯菊酯的抗药性为4.17,属低抗性;野外品系与敏感品系白纹伊蚊羧酸酯酶活性浓度分别为(1 019.768± 150.189) U/ml、(768.587±84.876)U/ml,野外品系酶活性明显高于敏感品系,差异有统计学意义(t=3.256,P=0.012).结论 深圳市坪山新区白纹伊蚊对氯菊酯的低抗性可能与羧酸酯酶酶活性水平高有关.

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

    Science.gov (United States)

    Yang, Dongfang; Wang, Xiliang; Chen, Yi-Tzai; Deng, Ruitang; Yan, Bingfang

    2009-05-15

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

  15. Domain Modeling: NP_001020366.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_001020366.1 chr16 Crystal structure of human carboxylesterase in complex with Co...enzyme A c2h7cf_ chr16/NP_001020366.1/NP_001020366.1_holo_23-554.pdb blast 35L,80N,82T,83S,91D,93K,94A,95G,9

  16. Quantitative Structure-Activity Relationships for Organophosphate Enzyme Inhibition (Briefing Charts)

    Science.gov (United States)

    2011-09-22

    Cleft Muscarinic/Nicotinic Receptor Cholinergic Nervous System “Normal Mechanism of Action” Citrate Pyruvate Acetyl CoA + + 6 Cholinergic...Choline Carrier Synaptic Cleft Muscarinic/Nicotinic Receptor CoA + + Acetylcholinesterase Hyperstimulation Citrate Pyruvate 7 Physiologically...neurotoxicity FAAH, CB1 Cannabinoid interactions AFMID teratogenesis APH neuropeptide metabolism Carboxylesterases Amidases Toxicity

  17. Joint acute toxicity of esfenvalerate and diazinon to larval fathead minnows (Pimephales promelas).

    Science.gov (United States)

    Denton, Debra L; Wheelock, Craig E; Murray, Shauna A; Deanovic, Linda A; Hammock, Bruce D; Hinton, David E

    2003-02-01

    California (USA) agriculture employs pyrethroid and organophosphate insecticides to control insects in orchards and other crops. Diazinon and esfenvalerate were selected for this study because of their application overlaps. Toxicological and biochemical responses of larval fathead minnows (Pimephales promelas) exposed singly and in combinations to esfenvalerate and diazinon were determined. Exposures were 96-h static renewal tests that used standard U.S. Environmental Protection Agency acute toxicity test methods. After pesticide exposures, larvae were evaluated for carboxylesterase and acetylcholinesterase activity, and histopathological effects. Carboxylesterase activity was examined because of its potential influence on the toxicity of both organophosphates and pyrethroids. In vivo studies demonstrated that diazinon significantly inhibited carboxylesterase activity at nominal water concentrations as low as 50 microg/L. However, esfenvalerate did not affect carboxylesterase activity at any concentration tested. Liver glycogen depletion was the only histopathological effect observed; this effect was demonstrated with the individual pesticides and pesticide combinations (i.e., mixtures). The combinations of diazinon and esfenvalerate causing acute toxicity to fathead minnow larvae appeared to be greater than additive (i.e., synergistic) in all three tests.

  18. Dicty_cDB: Contig-U07718-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 19 ) RecName: Full=Liver carboxylesterase; EC=3.1.1... 58 7e-11 AB201554_1( AB201554 |pid:none) Harmonia axy...r bra... 50 7e-10 EU783917_1( EU783917 |pid:none) Harmonia axyridis carboxylester

  19. Dicty_cDB: Contig-U16282-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ) Desulfococcus oleovorans Hxd3, ... 155 5e-36 AB201554_1( AB201554 |pid:none) Harmonia axyridis HaJHE mRNA ...sor -... 152 5e-35 EU783917_1( EU783917 |pid:none) Harmonia axyridis carboxylesterase... 152 5e-35 EU130462_

  20. Dicty_cDB: Contig-U09620-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available -19 AB201554_1( AB201554 |pid:none) Harmonia axyridis HaJHE mRNA for j... 96 2e-18 AE013599_2306( AE013599 |...3917_1( EU783917 |pid:none) Harmonia axyridis carboxylesterase... 94 1e-17 AJ8502

  1. Unidirectional cross-tolerance between the carbamate insecticide propoxur and the organophosphate disulfoton in mice.

    Science.gov (United States)

    Costa, L G; Murphy, S D

    1983-01-01

    Previous studies have shown that subchronic treatment of mice with the organophosphate insecticide, disulfoton, or the carbamate insecticide, propoxur, leads to the development of tolerance to their toxicity. Tolerance to disulfoton was due to a decrease in the number of muscarinic cholinergic receptors, while tolerance to propoxur appeared to be due to an induction of hepatic microsomal enzymes. In the present study we investigated if cross-tolerance between disulfoton and propoxur would occur. Cross-tolerance was evaluated by measuring acute toxicities, cholinesterase and carboxylesterase inhibition and hypothermic and antinociceptive effects. Mice tolerant to propoxur were cross-tolerant to the hypothermic and anticholinesterase effects of disulfoton. Similarly, when mice were pretreated with the microsomal enzyme inducer, phenobarbital, the toxicity of disulfoton was decreased. Mice made tolerant to disulfoton were cross-tolerant to the organophosphate chlorpyrifos, but were more sensitive than controls to the toxicity of propoxur. The acute toxicity of the organophosphate malathion was also increased in disulfoton-tolerant mice. Propoxur is metabolized by mixed function oxidases and possibly by a carboxylesterase. While hepatic microsomal enzymes appeared to be unchanged in disulfoton-tolerant mice, brain and liver carboxylesterase activities were significantly inhibited. Pretreatment of mice with the specific carboxylesterase inhibitor triorthotolylphosphate is known to greatly potentiate the toxicity of malathion and also potentiated, to a lesser extent, the toxicity of propoxur.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Esterase isozymes patterns of grape vine (Vitis vinifera L. are altered in response to fungicide exposure

    Directory of Open Access Journals (Sweden)

    Gleice Ribeiro Orasmo

    2015-10-01

    Full Text Available Current analysis characterizes the effect of different fungicides often applied for pest control on a-and b-esterase patterns of four economically important table-wine grape cultivars (Italia, Rubi, Benitaka and Brasil of Vitis vinifera. The a- and b-esterase patterns in bud leaves of the cultivars were assessed by native PAGE analysis. Cabrio Top® compound inhibited Est-2, Est-5, Est-6, Est-7, Est-8, Est-9 and Est-10 carboxylesterases, whereas Est-4, Est-11, Est-12, Est-13, Est-14 acetylesterases and Est-16 carboxylesterase were detected as weakly stained bands. Carboxylesterases and acetylesterases were also detected as weakly stained bands when exposed to fungicides Orthocide 500®, Positron Duo® and Folicur PM®. No changes in a- and b-esterase patterns were reported when the vines were exposed to the fungicides Rovral SC®, Kumulus DF®, Curzate M®, Score® or Cuprogarb 500®. The evidence of functional changes in carboxylesterase and acetylesterase levels in current study is a warning to grape producers on the dangers inherent in the indiscriminate use of potent and modern fungicides extensively used in agriculture. The inhibition effect of fungicides on esterase isozyme molecules seems to be independent of the fungicide chemical.

  3. Nerve Agent Hydrolysis Activity Designed into a Human Drug Metabolism Enzyme

    Science.gov (United States)

    2011-03-18

    carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun . Biochemistry 46: 5063–5071. 8. Hemmert AC, Otto TC, Wierdl M, Edwards CC...sarin, tabun , and cyclohexyl methylphosphonate-modified human butyrylcholinesterase. Chem Res Toxicol 22: 1680–1688. 14. Lenz DE, Broomfield CA, Yeung

  4. Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

    Science.gov (United States)

    Wheelock, C.E.; Eder, K.J.; Werner, I.; Huang, H.; Jones, P.D.; Brammell, B.F.; Elskus, A.A.; Hammock, B.D.

    2005-01-01

    Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 ??g/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 ??g/l), but not a low dose (1.2 ??g/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ???30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was

  5. Correlation between biochemical parameters and susceptibility of freshwater fish to malathion.

    Science.gov (United States)

    Li Shao-Non; Fan De-Fang

    1996-07-01

    Acute toxicity (96-h LC50) of malathion was tested using five species of freshwater fish, namely, topmouth gudgeon (Pseudorasbora parva), goldfish (Carassius auratus), nile tilapia (Tilapia nilotica), mosquitofish (Gambusia affinis), and rainbow trout (Salmo gairdneri). Correlation was found between susceptibility and biochemical parameters such as activity of brain acetylcholinesterase (AChE) and in vitro resistance of the enzyme to inhibition (IC50) of malaoxon (a major metabolite of malathion). The in vitro study also showed that malaoxon instead of malathion was the main inhibitor of AChE. Susceptibility to malathion was considerably changed as the fish was pretreated with piperonyl butoxide (PB, a P-450 inhibitor) and triphenyl phosphate (TPP, an inhibitor of carboxylesterase), respectively. Toxicity of malathion was significantly increased by TPP, but the responses of fish to PB were quite different among species. This suggested that both carboxylesterase and monooxygenase played an important role in susceptibility determination, and great variations existed among species in activity of monooxygenase.

  6. Investigation of the metabolism of rufinamide and its interaction with valproate.

    Science.gov (United States)

    Williams, Eric T; Carlson, J Eric; Lai, W George; Wong, Y Nancy; Yoshimura, Tsutomu; Critchley, David J; Narurkar, Milind

    2011-12-01

    Rufinamide was evaluated in vitro to determine which enzyme(s) are responsible for rufinamide hydrolysis and whether valproate, one of its metabolites (valproyl-CoA), and/or the rufinamide hydrolysis product (CGP 47292) could inhibit hydrolysis. Rufinamide hydrolysis was mediated primarily by human carboxylesterase (hCE) 1 and was nonsaturable up to 500 μM. Two-thirds of rufinamide hydrolysis was estimated to occur in human microsomes and one-third in cytosol. Valproate was a selective inhibitor for hCE1 compared to hCE2 and inhibition had a greater impact on rufinamide hydrolysis in microsomes than in cytosol. Valproyl-CoA caused similar inhibition of rufinamide hydrolysis in both microsomes and cytosol. Carboxylesterases were not significantly inhibited by CGP 47292. Inhibition of in vitro rufinamide hydrolysis by valproate could offer an explanation for the observed in vivo drug-drug interaction between the two antiepileptic drugs.

  7. Blood group and protein polymorphism gene frequencies for the andalusian horse breed: a comparison with four american horse breeds

    OpenAIRE

    Aguilar Sánchez, P.; Rodríguez-Gallardo, P.P.; Andrés Cara, D.F. de; J.L Vega-Pla

    1992-01-01

    Gene frecuencies at seventeen blood group and protein polymorphism loci for the andalusian horse breed are given. Standard methods of starch and polyacrylamide gel electrophoresis were used to identify inherited variants at the following enzyme and other protein loci: albumin (Al), transferrin (Tf), carboxylesterase (Es), A1B glycoprotein (Xk), vitamin D binding protein (Gc), protease inhibitor (Pi), 6-phosphogluconate dehydrogenase (PGD), phosphoglucomutase (PGM) and glucosephosphate isomera...

  8. Acetylation serves as a protective group in noscapine biosynthesis in opium poppy.

    Science.gov (United States)

    Dang, Thu-Thuy T; Chen, Xue; Facchini, Peter J

    2015-02-01

    We have characterized four sequential enzymes that transform 1-hydroxy-N-methylcanadine to narcotoline hemiacetal, completing our elucidation of noscapine biosynthesis in opium poppy. Two cytochromes P450 catalyze hydroxylations at C13 and C8 on the protoberberine scaffold, the latter step inducing ring opening and the formation of an aldehyde moiety. Acetylation at C13 before C8 hydroxylation introduces a protective group subsequently hydrolyzed by a carboxylesterase, which triggers rearrangement to a cyclic hemiacetal.

  9. Dicty_cDB: Contig-U15462-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ter chromos... 56 7e-07 EU130461_1( EU130461 |pid:none) Tetranychus cinnabarinus esterase ... 56 7e-07 AP006...riseus subsp. gri... 54 2e-06 AY790343_1( AY790343 |pid:none) Tetranychus cinnabari...) Bos taurus carboxylesterase 2 (int... 54 2e-06 EU130462_1( EU130462 |pid:none) Tetranychus cinnabari

  10. Effect of pretreatment with hepatic microsomal enzyme inducers on the toxicity of diazinon in calves.

    Science.gov (United States)

    Abdelsalam, E B; Ford, E J

    1986-11-01

    The pretreatment of calves with a single dose of 10 mg kg-1 dieldrin or 21 daily doses of 10 mg kg-1 phenobarbitone increased the toxicity of diazinon as reflected by the development of more severe clinical signs and greater depression in whole blood cholinesterase activity in the pretreated calves. Induction by dieldrin or phenobarbitone of the hepatic microsomal enzyme amidopyrine-N-demethylase was also accompanied by a concurrent rise in the liver carboxylesterase activity.

  11. Dicty_cDB: Contig-U10627-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available :none) Bos taurus carboxylesterase 2 (int... 43 0.007 DQ278875_1( DQ278875 |pid:none) Danio rerio thyroglobulin...|pid:none) Homo sapiens mRNA for Thyroglobuli... 38 0.23 AF080484_1( AF080484 |pid:none) Homo sapiens thyroglobulin... X02318_1( X02318 |pid:none) Rat mRNA fragment for thyroglobulin. 35 1.9 AF221622_1( AF221622 |pid:none) Rat

  12. Reversal of Zolpidem Intoxication By Sublingual Flumazenil

    Science.gov (United States)

    2008-11-01

    metabolized by the liver forming two inactive metabolites that are excreted in the urine . It is primarily hydrolyzed by a liver carboxylesterase...evaluated in the study. Women who were pregnant or attempting to become pregnant were excluded. Female participants were administered a urine ...RD (1995, March). The use of amphetamines in U.S. Air Force tactical operations during Desert Shield and Storm. Aviation, Space, and Environmental

  13. The Anopheles gambiae detoxification chip: A highly specific microarray to study metabolic-based insecticide resistance in malaria vectors

    OpenAIRE

    2005-01-01

    Metabolic pathways play an important role in insecticide resistance, but the full spectra of the genes involved in resistance has not been established. We constructed a microarray containing unique fragments from 230 Anopheles gambiae genes putatively involved in insecticide metabolism [cytochrome P450s (P450s), GSTs, and carboxylesterases and redox genes, partners of the P450 oxidative metabolic complex, and various controls]. We used this detox chip to monitor the expression of the detoxify...

  14. Enantiospecific determination of dl-methylphenidate and dl-ethylphenidate in plasma by liquid chromatography-tandem mass spectrometry: Application to human ethanol interactions

    OpenAIRE

    Zhu, Hao-Jie; Patrick, Kennerly S.; Markowitz, S

    2011-01-01

    In humans, concomitant dl-methylphenidate (dl-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite dl-ethylphenidate (dl-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between d- and l-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties....

  15. B-type esterases in the snail Xeropicta derbentina: An enzymological analysis to evaluate their use as biomarkers of pesticide exposure

    Energy Technology Data Exchange (ETDEWEB)

    Laguerre, Christel [Universite d' Avignon et des Pays de Vaucluse, UMR 406 UAPV/INRA, F-84914 Avignon (France); INRA, Laboratoire de Toxicologie Environnementale, UMR 406 UAPV/INRA, F-84914 Avignon (France); Sanchez-Hernandez, Juan C. [Laboratory of Ecotoxicology, Faculty of Environmental Science, University of Castilla-La Mancha, Avda. Carlos III s/n, 45071 Toledo (Spain); Koehler, Heinz R. [Animal Physiological Ecology, University of Tuebingen, Konrad-Adenauer-Strasse 20, D-72072 Tuebingen (Germany); Triebskorn, Rita [Animal Physiological Ecology, University of Tuebingen, Konrad-Adenauer-Strasse 20, D-72072 Tuebingen (Germany); Steinbeis-Transfer Center for Ecotoxicology and Ecophysiology, Blumenstrasse 13, D-72108 Rottenburg (Germany); Capowiez, Yvan [INRA, Unite PSH, F- 84914 Avignon (France); Rault, Magali [Universite d' Avignon et des Pays de Vaucluse, UMR 406 UAPV/INRA, F-84914 Avignon (France); INRA, Laboratoire de Toxicologie Environnementale, UMR 406 UAPV/INRA, F-84914 Avignon (France); Mazzia, Christophe [Universite d' Avignon et des Pays de Vaucluse, UMR 406 UAPV/INRA, F-84914 Avignon (France); INRA, Laboratoire de Toxicologie Environnementale, UMR 406 UAPV/INRA, F-84914 Avignon (France)], E-mail: mazzia@avignon.inra.fr

    2009-01-15

    The study was prompted to characterize the B-type esterase activities in the terrestrial snail Xeropicta derbentina and to evaluate its sensitivity to organophosphorus and carbamate pesticides. Specific cholinesterase and carboxylesterase activities were mainly obtained with acetylthiocholine (K{sub m} = 77.2 mM; V{sub max} = 38.2 mU/mg protein) and 1-naphthyl acetate (K{sub m} = 222 mM, V{sub max} = 1095 mU/mg protein) substrates, respectively. Acetylcholinesterase activity was concentration-dependently inhibited by chlorpyrifos-oxon, dichlorvos, carbaryl and carbofuran (IC50 = 1.35 x 10{sup -5}-3.80 x 10{sup -8} M). The organophosphate-inhibited acetylcholinesterase activity was reactivated in the presence of pyridine-2-aldoxime methochloride. Carboxylesterase activity was inhibited by organophosphorus insecticides (IC50 = 1.20 x 10{sup -5}-2.98 x 10{sup -8} M) but not by carbamates. B-esterase-specific differences in the inhibition by organophosphates and carbamates are discussed with respect to the buffering capacity of the carboxylesterase to reduce pesticide toxicity. These results suggest that B-type esterases in X. derbentina are suitable biomarkers of pesticide exposure and that this snail could be used as sentinel species in field monitoring of Mediterranean climate regions. - Characterization of the B-type esterases in the terrestrial snail Xeropicta derbentina in order to evaluate pesticide exposure.

  16. Correlation between pesticide resistance and enzyme activity in the diamondback moth, Plutella xylostella.

    Science.gov (United States)

    Gong, Ya-Jun; Wang, Ze-Hua; Shi, Bao-Cai; Kang, Zong-Jiang; Zhu, Liang; Jin, Gui-Hua; Weig, Shu-Jun

    2013-01-01

    The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most important pests that has developed high pesticide resistance. The resistances of five Chinese populations of this moth, four resistant strains (from Beijing, Henan, Fujian, and Guangdong) and one susceptible strain, to five pesticides were determined, and the activities of carboxylesterase, glutathione S-transferase, and acetylcholine esterase were tested in all five populations. The correlations between pesticide resistance and enzyme activity were analyzed. The results showed that the resistance status to the five pesticides was different among the five populations. The resistance ratios of the Beijing and Henan populations to spinosad were 5.84 and 8.22, respectively, and those to beta-cypermethrin were 4.91 and 4.98, respectively. These ratios were higher than those for the Fujian and Guangdong populations. The Fujian population was more sensitive to abamectin and chlorpyrifos than the susceptible population (the resistance ratios were 0.14 and 0.91, respectively); in fact, the median lethal concentration for P. xylostella was significantly higher for chlorpyrifos than that for any of the other four pesticides. The carboxylesterase activity in P. xylostella showed positive correlations with the resistance to spinosad, beta-cypermethrin, chlorpyrifos, and abamectin, but no correlation was observed between the carboxylesterase activity and resistance to emamectin benzoate, between glutathione S-transferase activity and resistance to any of the five pesticides tested, or between acetylcholine esterase activity and any of the pesticides except for emamectin benzoate.

  17. Species-specific differences in biomarker responses in two ecologically different earthworms exposed to the insecticide dimethoate.

    Science.gov (United States)

    Velki, Mirna; Hackenberger, Branimir K

    2012-08-01

    Earthworms ingest large amounts of soil and therefore are continuously exposed to contaminants through their alimentary surfaces. Additionally, several studies have shown that earthworm skin is a significant route of contaminant uptake as well. In order to determine effects of dimethoate, a broad-spectrum organophosphorous insecticide, two ecologically different earthworm species were used - Eisenia andrei and Octolasion lacteum. Although several studies used soil organisms to investigate the effects of dimethoate, none of these studies included investigations of dimethoate effects on biochemical biomarkers in earthworms. Earthworms were exposed to 0.001, 0.005, 0.01, 0.5 and 1 μg/cm(2) of dimethoate for 24 h, and the activities of acetylcholinesterase, carboxylesterase, catalase and efflux pump were measured. In both earthworm species dimethoate caused significant inhibition of acetylcholinesterase and carboxylesterase activities, however in E. andrei an hormetic effect was evident. Efflux pump activity was inhibited only in E. andrei, and catalase activity was significantly inhibited in both earthworm species. Additionally, responses of earthworm acetylcholinesterase, carboxylesterase and catalase activity to dimethoate were examined through in vitro experiments. Comparison of responses between E. andrei and O. lacteum has shown significant differences, and E. andrei has proved to be less susceptible to dimethoate exposure.

  18. The resistant mechanism of Myzus persicae to pesticide in Yunnan Province%云南烟蚜抗药性机制研究

    Institute of Scientific and Technical Information of China (English)

    宋春满; 邓建华

    2012-01-01

    通过比较云南烟蚜敏感品系和抗性品系的解毒酶(α-乙酸萘酯羧酸酯酶、β-乙酸萘酯羧酸酯酶)和靶标酶(乙酰胆碱酯酶)的活力,研究了烟蚜对有机磷、拟除虫菊酯和氨基甲酸酯类杀虫剂抗性的生化机制,并通过酯酶基因扩增检测和钠离子通道突变检测,研究了其抗性的分子机制.结果表明:α-乙酸萘酯羧酸酯酶活力增强是烟蚜对有机磷类、氨基甲酸酯类杀虫剂及拟除虫菊酯类杀虫剂的抗性机制之一;乙酰胆碱酯酶在烟蚜对有机磷杀虫剂抗性中起重要作用;3个抗性品系烟蚜均没有发生酯酶基因扩增,抗拟除虫菊酯品系烟蚜发生了钠离子通道突变.%To study the biochemical and molecular mechanism of resistance to organophosphorus, pyre- throid and carbarmate pesticide, the activity of the detoxicating enzymes (α - NA carboxylesterase, β — NA carboxylesterase) and the target enzyme ( acetylcholinesterase) were compared among the susceptible and the resistant strain of Myzus persicae. The results showed that enhanced α - NA carboxylesterase played an important role in the resistance to the three kinds of pesticides, so did the acetylcholinesterase to organophosphorus pesticide. All the three resistant strains had no amplified esterase genes, and the strain resistant to pyrethroid had a mutation in sodium channel.

  19. [Resistance mechanisms of Spodoptera exigua (Hübner) to fenvalerate and alpha-cypermethrin].

    Science.gov (United States)

    Lan, Yi-Quan; Zhao, Shi-Xi

    2010-01-01

    By using synergist bioassay and biochemical analysis, this paper approached the resistance mechanisms of Spodoptera exigua (Hübner) to fenvalerate and alpha-cypermethrin. The synergistic ratios of piperonyl butoxide (PBO), o, o-diethyl-o-phenyl-thiophosphate (SV1), triphenyl phosphate (TPP), and diethyl maleate (DEM) between fenvalerate-resistant strain (Fen-R) and susceptible strain were 10.2, 7.8, 12.5, and 1.1, and those between alpha-cypermethrin resistant strain (Cyp-R) and susceptible strain were 21.6, 15.5, 8.6, and 1.2, respectively. Significant synergisms of PBO, SV1, and TPP to fenvalerate and alpha-cypermethrin were observed, implying that multifunctional oxidase and carboxylesterase were involved in the resistance to fenvalerate and alpha-cypermethrin. The carboxylesterase activities in the fourth instar larvae of Cyp-R and Fen-R strains were 1.9 and 2.2 folds of the corresponding susceptible strains, respectively, but no differences were found in the glutathione-S-transferase activities between the resistant and susceptible strains, which indicated that carboxylesterase played an important role in the resistance of S. exigua to fenvalerate and alpha-cypermethrin, while glutathione-S-transferase contributed little to the resistance. There were no significant differences in the Na-K-ATPase activities between the resistant and susceptible strains, but the inhibition of fenvalerate and alpha-cypermethrin on Na-K-ATPase was higher in the susceptible strains than in the resistant strains, indicating the decreased sensitivity of Na-K-ATPase in resistant strains.

  20. Protection against Acetylcholinesterase Inhibitor Toxicity by Alpha- Adrenergic Agonists

    Science.gov (United States)

    1992-10-28

    in Rats 22 " fable 4 Prevalence of Soman-Evoked Behaviors at the Time of Maximal Expression 23 Table 5 Open-field Motor Activity 2 Days After Soman... Brecht , K.M. and Lenz, D.E. (1987). Effect of carboxylesterase inhibition on carbamate protection against soman toxicity. In: Proceedings of the 1987...Medical Defense Bioscience Review, Aberdeen Proving Ground. pp. 17-24. 33. Maxwell, D.M., Brecht . K.M. and O’neill, B.L1 (1987).The effect of

  1. 东亚飞蝗羧酸酯酶基因的克隆和原核表达%Cloning and prokaryotical expression of CarE gene in Locusta migratoria manilensis

    Institute of Scientific and Technical Information of China (English)

    李清; 张学尧; 张建珍; 马恩波

    2012-01-01

    羧酸酯酶是昆虫体内重要的代谢解毒酶系,其主要功能是水解和结合内源性和外源性含有酯键的有毒物质,减缓其到达靶标部位的时间.东亚飞蝗Locusta migratoria manilensis (Meyen)是我国重要的农业害虫,对其羧酸酯酶基因克隆和表达有助于深入探索杀虫剂代谢毒理机制.本研究首先对羧酸酯酶基因(CarE4)进行了克隆,并将其插入到pCold TF DNA Vector中,在大肠杆菌中进行了原核表达,最后用疏水层析和离子交换层析方法对目的蛋白进行了纯化.本文成功建立了羧酸酯酶蛋白原核表达和纯化技术体系,为进一步研究东亚飞蝗羧酸酯酶的生理功能、结构特点和作用原理提供了基础资料.%The migratory locust, Locusta migratoria manilensis (Meyen) ( Orthoptera, Acridoidea) , is one of the most important pests in China. Carboxylesterase is an important metabolic detoxification enzyme in insects the main function of which is the hydrolysis of endogenous and exogenous toxic substances containing ester bonds. In this study, the carboxylesterase gene (CarE4) wa9 cloned and inserted into the pCold TF DNA vector. A recombinant carboxylesterase was successfully expressed in E. coli and the recombinant protein purified by hydrophobic chromatography and ion exchange chromatography. The results provide a basis for further study of the physiological function, structural characteristics and function of locust carboxylesterases.

  2. A comparative study on esterases from three species of Raillietina.

    Science.gov (United States)

    Balasubramanian, M P; Dhandayuthapani, S; Nellaiappan, K; Ramalingam, K

    1984-06-01

    The multiplicity of soluble esterases in Raillietina tetragona, R. echinobothrida and R. cesticillus was studied by use of slab polyacrylamide gel electrophoresis. Five fractions of esterase activity were observed in R. tetragona, seven in R. echinobothrida and three in R. cesticillus. The various fractions of esterase activity of closely related species of Raillietina showed differential behaviour towards various chemicals. Based on the inhibitory effect of inhibitors p-CMB, EDTA, malathion, silver nitrate and eserine sulphate, the various esterases have been classified into arylesterase, carboxylesterase, acetylesterase and cholinesterase.

  3. Complete genome of the potential thermozyme producer Anoxybacillus gonensis G2(T) isolated from the Gönen hot springs in Turkey.

    Science.gov (United States)

    Lim, Yan Lue; Chan, Kok-Gan; Ee, Robson; Belduz, Ali Osman; Canakci, Sabriye; Kahar, Ummirul Mukminin; Yaakop, Amira Suriaty; Goh, Kian Mau

    2015-10-20

    Anoxybacillus gonensis type strain G2(T) (=NCIMB 13,933(T) =NCCB 100040(T)) has been isolated from the Gönen hot springs in Turkey. This strain produces a number of well-studied, biotechnologically important enzymes, including xylose isomerase, carboxylesterase, and fructose-1,6-bisphosphate aldolase. In addition, this strain is an excellent candidate for the bioremediation of areas with heavy metal pollution. Here, we present a high-quality, annotated, complete genome of A. gonensis G2(T). Furthermore, this report provides insights into several novel enzymes of strain G2(T) and their potential industrial applications.

  4. Investigating the impact of missense mutations in hCES1 by in silico structure-based approaches

    DEFF Research Database (Denmark)

    Nzabonimpa, Grace Shema; Rasmussen, Henrik Berg; Brunak, Søren

    2016-01-01

    Genetic variations in drug-metabolizing enzymes have been reported to influence pharmacokinetics, drug dosage and other aspects that affect therapeutic outcomes. Most particularly, non-synonymous single-nucleotide polymorphisms (nsSNPs) resulting in amino acid changes disrupt potential functional...... level. Examples of in silico studies of carboxylesterases (CESs) are discussed, ranging from exploring the effect of mutations on enzyme activity to predicting the metabolism of new hCES1 substrates as well as to guiding rational design of CES-selective inhibitors....

  5. Toxicology and Biodistribution Studies for MGH2.1, an Oncolytic Virus that Expresses Two Prodrug-activating Genes, in Combination with Prodrugs

    OpenAIRE

    Kasai, Kazue; Nakashima, Hiroshi; Liu, Fang; Kerr, Samantha; Wang, Jiang, 1959-; Phelps, Mitch; Potter, Philip M.; Goins, William B; Fernandez, Soledad A.; Chiocca, E. Antonio

    2013-01-01

    MGH2.1 is a herpes simplex virus type 1 (HSV1) oncolytic virus that expresses two prodrug-activating transgenes: the cyclophosphamide (CPA)-activating cytochrome P4502B1 (CYP2B1) and the CPT11-activating secreted human intestinal carboxylesterase (shiCE). Toxicology and biodistribution of MGH2.1 in the presence/absence of prodrugs was evaluated in mice. MGH2.1 ± prodrugs was cytotoxic to human glioma cells, but not to normal cells. Pharmacokinetically, intracranial MGH2.1 did not significantl...

  6. [Effects of alcohol extracts from three kinds of biomass energy plant tissues on biological activity of Bemisia tabaci].

    Science.gov (United States)

    Zhou, Fu-cai; Zhou, Gui-sheng; Li, Chuan-ming; Yang, Yi-zhong; Qin, Pei

    2009-03-01

    To test the feasibility of using raw extracts from the tissues of biomass energy plants Ricinus communi and Kosteletzkya virginica as plant protection agents, the alcohol extracts from R. communi seed and leaf and from K. virginica leaf were used to treat adult Bemisia tabaci by spraying. The glutathione S-transferase and carboxylesterase activities in B. tabaci body were measured after treated for 4 h, 24 h, 48 h, 72 h, and 96 h, and the olfaction responses of B. tabaci to the alcohol extracts were detected with a Y-tube olfactomet. All the three alcohol extracts obviously inhibited the glutathione S-transferase and carboxylesterase activities in a concentration-dependent manner. The inhibitory effect of the 250-times diluted alcohol extracts on the two enzyme activities was equivalent to that of 3000 times-diluted 1.8% avermectins. In addition, the 250-times diluted alcohol extracts had obvious repellent effect on B. tabaci, with the repellent coefficient of the alcohol extracts from R. communi seed and leaf and from K, virginica leaf being 100.0%, 96.7%, and 79.4%, respectively. All of these suggested that the test three alcohol extracts had repellent and other biological effects on B. tabaci.

  7. Analysis of thermal adaptation in the HSL enzyme family.

    Science.gov (United States)

    Mandrich, L; Pezzullo, M; Del Vecchio, P; Barone, G; Rossi, M; Manco, G

    2004-01-01

    The recently solved three-dimensional (3D) structures of two thermostable members of the carboxylesterase/lipase HSL family, namely the Alicyclobacillus (formerly Bacillus) acidocaldarius and Archaeoglobus fulgidus carboxylesterases (EST2 and AFEST, respectively) were compared with that of the mesophilic homologous counterpart Brefeldine A esterase from Bacillus subtilis. Since the 3D homology models of other members of the HSL family were also available, we performed a structural alignment with all these sequences. The resulting alignment was used to assess the amino acid "traffic rule" in the HSL family. Quite surprisingly, the data were in very good agreement with those recently reported from two independent groups and based on the comparison of a huge number of homologous sequences from the genus Bacillus, Methanococcus and Deinococcus/Thermus. Taken as a whole, the data point to the statistical meaning of defined amino acid conversions going from psychrophilic to hyperthermophilic sequences. We identified and mapped several such changes onto the EST2 structure and observed that such mutations were localized mostly in loops regions or alpha-helices and were mostly excluded from the active site. A site-directed mutagenesis of two of the identified residues confirmed they were involved in thermal stability.

  8. Combined neonicotinoid pesticide and parasite stress alter honeybee queens’ physiology and survival

    Science.gov (United States)

    Dussaubat, Claudia; Maisonnasse, Alban; Crauser, Didier; Tchamitchian, Sylvie; Bonnet, Marc; Cousin, Marianne; Kretzschmar, André; Brunet, Jean-Luc; Le Conte, Yves

    2016-01-01

    Honeybee colony survival strongly relies on the queen to overcome worker losses exposed to combined stressors like pesticides and parasites. Queen’s capacity to withstand these stressors is however very little known. The effects of the common neonicotinoid pesticide imidacloprid in a chronic and sublethal exposure together with the wide distributed parasite Nosema ceranae have therefore been investigated on queen’s physiology and survivorship in laboratory and field conditions. Early physiological changes were observed on queens, particularly the increase of enzyme activities (catalase [CAT] and glutathione-S-transferase [GST] in the heads) related to protective responses to xenobiotics and oxidative stress against pesticide and parasite alone or combined. Stressors also alter the activity of two other enzymes (carboxylesterase alpha [CaE α] and carboxylesterase para [CaE p] in the midguts) involved in metabolic and detoxification functions. Furthermore, single and combined effects of pesticide and parasite decrease survivorship of queens introduced into mating hives for three months. Because colony demographic regulation relies on queen’s fertility, the compromise of its physiology and life can seriously menace colony survival under pressure of combined stressors. PMID:27578396

  9. Combined neonicotinoid pesticide and parasite stress alter honeybee queens' physiology and survival.

    Science.gov (United States)

    Dussaubat, Claudia; Maisonnasse, Alban; Crauser, Didier; Tchamitchian, Sylvie; Bonnet, Marc; Cousin, Marianne; Kretzschmar, André; Brunet, Jean-Luc; Le Conte, Yves

    2016-01-01

    Honeybee colony survival strongly relies on the queen to overcome worker losses exposed to combined stressors like pesticides and parasites. Queen's capacity to withstand these stressors is however very little known. The effects of the common neonicotinoid pesticide imidacloprid in a chronic and sublethal exposure together with the wide distributed parasite Nosema ceranae have therefore been investigated on queen's physiology and survivorship in laboratory and field conditions. Early physiological changes were observed on queens, particularly the increase of enzyme activities (catalase [CAT] and glutathione-S-transferase [GST] in the heads) related to protective responses to xenobiotics and oxidative stress against pesticide and parasite alone or combined. Stressors also alter the activity of two other enzymes (carboxylesterase alpha [CaE α] and carboxylesterase para [CaE p] in the midguts) involved in metabolic and detoxification functions. Furthermore, single and combined effects of pesticide and parasite decrease survivorship of queens introduced into mating hives for three months. Because colony demographic regulation relies on queen's fertility, the compromise of its physiology and life can seriously menace colony survival under pressure of combined stressors.

  10. Evidence for diazinon-mediated inhibition of cis-permethrin metabolism and its effects on reproductive toxicity in adult male mice.

    Science.gov (United States)

    Wang, Dong; Kamijima, Michihiro; Okamura, Ai; Ito, Yuki; Yanagiba, Yukie; Jia, Xiao-fang; Naito, Hisao; Ueyama, Jun; Nakajima, Tamie

    2012-12-01

    The potential toxicity resulting from combinatorial effects of organophosphorus and pyrethroid insecticides are not completely known. We evaluated male reproductive toxicity in mice co-exposed to diazinon and cis-permethrin. Nine-week-old male Sv/129 mice were exposed to diazinon (10 μmol/kg/day) or cis-permethrin (90 μmol/kg/day) alone or in combination (100 μmol/kg/day), or vehicle (corn oil), for 6 weeks. Diazinon and the diazinon-permethrin mixture inhibited plasma and liver carboxylesterase activities. In the mixture group, urinary excretion of cis-permethrin metabolite 3-phenoxybenzoic acid decreased along with increased plasma and testicular concentrations of cis-permethrin, while excretion of diazinon metabolites, diethylphosphate and diethylthiophosphate, did not change, versus mice exposed to each chemical alone, which suggested that inhibition of carboxylesterase decreased the metabolic capacity to cis-permethrin. Though the co-exposure decreased testosterone biosynthesis, increased degenerate germ cells in seminiferous tubule and sperm morphological abnormalities versus controls more clearly than exposure to cis-permethrin alone, the expected potentiation of toxicity was not evident.

  11. Biochemical biomarkers in Oreochromis niloticus exposed to mixtures of benzo[a]pyrene and diazinon.

    Science.gov (United States)

    Pereira Trídico, Camila; Ferreira Rodrigues, Aline Cristina; Nogueira, Lilian; da Silva, Daniele Caetano; Benedito Moreira, Altair; de Almeida, Eduardo Alves

    2010-07-01

    Biochemical biomarkers (the activities of acetylcholinesterase, 7-ethoxyresorufin-O-deetilase, carboxylesterase, catalase, glutathione peroxidase and glutathione S-transferase) were evaluated in Nile tilapia (Oreochromis niloticus) that had been exposed to benzo[a]pyrene (BaP) and the organophosphate pesticide diazinon (DZ), at 0.5mg/L. The animals were pre-exposed to BaP for three days, and DZ was then added to both non-exposed and pre-exposed groups, being exposed for 2 and 7 additional days. The level of BaP was also measured in the bile. BaP caused the induction of phase I and II enzymes, and DZ caused carboxylesterase inhibition in gills but not in liver. AChE activity was unchanged. No significant modulation was observed in antioxidant enzymes. When in combination with BaP, DZ caused a significant decrease of EROD and GST induction. Levels of BaP in the bile were also increased in fish exposed to BaP combined with DZ, indicating an interference of DZ in responses activated by BaP.

  12. Biodegradation of malathion by Bacillus licheniformis strain ML-1

    Directory of Open Access Journals (Sweden)

    Khan Sara

    2016-01-01

    Full Text Available Malathion, a well-known organophosphate pesticide, has been used in agriculture over the last two decades for controlling pests of economically important crops. In the present study, a single bacterium, ML-1, was isolated by soil-enrichment technique and identified as Bacillus licheniformis on the basis of the 16S rRNA technique. The bacterium was grown in carbon-free minimal salt medium (MSM and was found to be very efficient in utilizing malathion as the sole source of carbon. Biodegradation experiments were performed in MSM without carbon source to determine the malathion degradation by the selected strain, and the residues of malathion were determined quantitatively using HPLC techniques. Bacillus licheniformis showed very promising results and efficiently consumed malathion as the sole carbon source via malathion carboxylesterase (MCE, and about 78% malathion was degraded within 5 days. The carboxylesterase activity was determined by using crude extract while using malathion as substrate, and the residues were determined by HPLC. It has been found that the MCE hydrolyzed 87% malathion within 96 h of incubation. Characterization of crude MCE revealed that the enzyme is robust in nature in terms of organic solvents, as it was found to be stable in various concentrations of ethanol and acetonitrile. Similarly, and it can work in a wide pH and temperature range. The results of this study highlighted the potential of Bacillus licheniformis strain ML-1 as a biodegrader that can be used for the bioremediation of malathion-contaminated soil.

  13. Trends in detoxification enzymes and heavy metal accumulation in ground beetles (Coleoptera: Carabidae) inhabiting a gradient of pollution.

    Science.gov (United States)

    Stone, David; Jepson, Paul; Laskowski, Ryszard

    2002-05-01

    Non-specfic carboxylesterase and glutathione S-transferase activity was measured in the ground beetle, Pterosthicus oblongopunctatus (Coleoptera: Carabidae), from five sites along a gradient of heavy metal pollution. A previous study determined that beetles from the two most polluted sites (site codes OLK2 and OLK3) were more susceptible to additional stressors compared with beetles from the reference site (Stone et al., Environ. Pollut. 113, 239-244 2001), suggesting the possibility of physiological impairment. Metal body burdens in ground beetles from five sites along the gradient ranged from 79 to 201 microg/g Zn, 0.174 to 8.66 microg/g Pb and 1.14 to 10.8 microg/g Cd, whereas Cu seemed to be efficiently regulated regardless of metal levels in the soil. Beetle mid- and hindguts were homogenized and the soluble fraction containing glutathione S-transferase (GST) and carboxylesterase (CaE) was assayed using kinetic analyses. Significantly higher levels of GST were found only in female beetles from the most polluted sites (OLK2 and OLK3; P=0.049, P<0.001, respectively) compared with the reference site (OLK7). In addition, OLK3 females had significantly higher levels of CaE compared with the reference beetles (P=0.01). Male beetles did not differ in enzyme activity along the metal gradient. Overall, obvious trends in detoxification enzymes were not detected in ground beetles in association with metal body burdens.

  14. Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor α and β and androgen receptor.

    Science.gov (United States)

    Watanabe, Yoko; Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Ohta, Shigeru; Kitamura, Shigeyuki

    2013-07-01

    The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor α (hERα), hERβ and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hERα and/or hERβ agonistic activity at concentrations of ≤ 1 × 10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C₁ to C₁₂, heptylparaben (C₇) and pentylparaben (C₅) showed the most potent ERα and ERβ agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C₁ or lengthened to C₁₂. Most parabens showing estrogenic activity exhibited ERβ-agonistic activity at lower concentrations than those inducing ERα-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ERβ over ERα; their interactions with ERα/β are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity.

  15. In vitro stability and metabolism of salvinorin A in rat plasma.

    Science.gov (United States)

    Tsujikawa, K; Kuwayama, K; Miyaguchi, H; Kanamori, T; Iwata, Y T; Inoue, H

    2009-05-01

    Salvinorin A is the main active psychoactive ingredient in Salvia divinorum, a Mexican plant that has been widely available as a hallucinogen in recent years. The aims of this study were to investigate the stability of salvinorin A in rat plasma, esterases responsible for its degradation, and estimation of the degradation products. The apparent first-order rate constants of salvinorin A at 37 degrees C, 25 degrees C, and 4 degrees C were 3.8 x 10(-1), 1.1 x 10(-1), and Salvinorin A degradation was markedly inhibited by the addition of sodium fluoride, an esterase inhibitor. Moreover, phenylmethylsulfonyl fluoride (serine esterase inhibitor) and bis-p-nitrophenylphosphate (carboxylesterase inhibitor) also inhibited salvinorin A degradation. In contrast, little or no suppression of the degradation was seen with 5,5'-dithiobis-2-nitrobenzoic acid (arylesterase inhibitor),ethopropazine (butyrylcholinesterase inhibitor), and BW284c51 (acetylcholineseterase inhibitor). These findings indicated that carboxylesterase was mainly involved in the salvinorin A hydrolysis in rat plasma.4. The degradation products of salvinorin A estimated by liquid chromatography-mass spectrometry included the deacetylated form (salvinorin B) and the lactone-ring-open forms of salvinorin A and salvinorin B. This lactone-ring-opening reactions were involved in calcium-dependent lactonase.

  16. Antioxidant and neuroprotective potential of chitooligomers in Caenorhabditis elegans exposed to Monocrotophos.

    Science.gov (United States)

    Nidheesh, T; Salim, Chinnu; Rajini, P S; Suresh, P V

    2016-01-01

    The objectives of this investigation were to establish the propensity of the chitooligomers (COS) to ameliorate neurodegeneration and oxidative stress in Caenorhabditis elegans induced by an organophosphorus insecticide, Monocrotophos (MCP). COS was prepared from α-chitosan by the enzymatic method using chitosanase and characterized by HPLC and electron spray ionization-TOF-(ESI-TOF)-MS. We exposed age synchronized L4 C. elegans worms (both wild type N2 and transgenic strain BZ555 (Pdat-1:GFP) to sublethal concentration of MCP (0.75mM) for 24h in the presence or absence of COS (0.2mM). The neuroprotective effect of COS was examined in N2 worms in terms of brood size, lifespan, egg laying, dopamine content, acetylcholinesterase and carboxylesterase activity and by direct visualization and quantification of degeneration of dopaminergic neurons in BZ555. Exposure to COS extended lifespan, normalized egg laying, increased brood size, decreased the dopaminergic neurodegeneration, increased the dopamine content and increased AChE and carboxylesterase activity in C. elegans treated with MCP. COS induced a significant decrease in reactive oxygen species and increased the reduced glutathione level as well as increased superoxide dismutase and catalase activity. Our findings demonstrate that COS significantly inhibits the dopaminergic neurodegeneration and associated physiological alterations induced by MCP in C. elegans by attenuating the oxidative stress as well.

  17. Tolerance to the carbamate insecticide propoxur.

    Science.gov (United States)

    Costa, L G; Hand, H; Schwab, B W; Murphy, S D

    1981-01-01

    Male mice were given the carbamate insecticide propoxur (2-isopropoxy phenyl methylcarbamate; Baygon) in the drinking water at weekly increasing concentrations (from 50 to 2000 ppm), for a period of 6 weeks. At the end of the treatment the LD50 for propoxur was significantly higher in the treated animals as compared with controls. Propoxur-treated animals were also resistant to the hypothermic effect of an acute administration of the same compound. Groups of mice were challenged with the cholinergic agonist carbachol at intervals during the drinking water dosing and at its end. No differences in sensitivity to carbachol acute toxicity were found between control and treated animals. Propoxur-tolerant animals were also not resistant to the hypothermic effect of oxotremorine, another cholinergic agonist. [3H]Quinuclidinyl benzilate ([3H]QNB) binding (a measure of muscarinic receptor density and affinity) in forebrain, hindbrain and ileum never differed in control and treated mice. The possibility that repeated administrations of propoxur induced increased metabolic inactivation was tested by measuring hexobarbital sleeping time and carboxylesterase activity in treated and control mice. No changes in tissue carboxylesterase activities occurred but hexobarbital sleeping time was significantly reduced in propoxur treated animals suggesting an induction of hepatic microsomal enzymes. These results suggest that tolerance to propoxur is not mediated by a decrease of cholinergic receptors, as reported for other acetylcholinesterase inhibitors, but possibly by an enhancement of its metabolism.

  18. Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases.

    Science.gov (United States)

    Johnson, Reed M; Wen, Zhimou; Schuler, Mary A; Berenbaum, May R

    2006-08-01

    Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.

  19. A COMPARISON OF THE ACTIVITY OF DETOXICATING ENZYMES AND TARGET ENZYME OFMYZUS PERSICAE IN DIFFERENT TOBACCO-PRODUCING AREAS OF YUNNAN%云南不同烟区烟蚜解毒酶与靶标酶活力的比较

    Institute of Scientific and Technical Information of China (English)

    宋春满; 李天飞; 邓建华; 高家合; 吴兴富

    2001-01-01

    The activity of the detoxicating enzymes related to pesticide resistan ce in Myzus persicae (α-NA carboxylesterase,β-NA carboxylesterase,glutathione transferase and acid phosphatase) and the target enzyme (acetylcholi nesterase) was determined. A comparison of the enzyme activity between the field populations of the pest in 7 major tobacco- producing areas in Yunnan revealed a highly significant difference in the enzyme activity of M. persicae in dif fere nt areas. The activity of all the 5 enzymes studied was highest in Maguohe popul ation of Qujing and lowest in the Xieyang population of Lijiang.%通过测定与烟蚜抗药性相关的解毒酶(α-NA羧酸酯酶、β-NA羧酸酯酶、谷胱甘肽转移酶和酸性磷酸酯酶)和靶标酶(乙酰胆碱酯酶)的活力,比较了云南7个主要烟区田间烟蚜种群的酶活力差异,发现不同烟区烟蚜酶活力差异极显著,5种酶的活力均以曲靖马过河种群最高,丽江斜阳种群最低。

  20. Temporal allocation of metabolic tolerance in the body of beet armyworm in response to three gossypol-cotton cultivars

    Institute of Scientific and Technical Information of China (English)

    Marvin; K; HARRIS

    2009-01-01

    The nutrient composition and enzyme activities in larvae of the beet armyworm, Spodoptera exigua (Hbner), fed on high, medium or low gossypol cotton cultivars were examined at different time intervals. Significantly lower free fatty acid was observed in larvae fed for 6 h on high gossypol ’M9101’ compared to larvae fed on the low (ZMS13) and intermediate (HZ401) gossypol cultivars. Significantly higher trypsin activity was observed in larvae fed on high gossypol ’M9101’ for 24 h compared to those fed for 1, 4 and 6 h. Significantly higher catalase and total superoxide dismutase enzyme activities were observed in larvae of S. exigua fed on high gossypol ’M9101’ compared with low gossypol cultivars ’ZMS13’ and ’HZ401’ for 1, 4, 6 and 24 h. However, significantly lower carboxylesterase and acetylcholinesterase enzyme activities were found in larvae fed on high gossypol ’M9101’ compared with the other cultivars for 1, 4, 6 and 24 h. The interaction between cotton variety and beet armyworm infestation time significantly affected the carboxylesterase enzyme activity in S. exigua. The characterization of the effects of plant allelochemicals on herbivorous larvae is important for aiding understanding of plant-insect interaction as well as in devising solutions to pest problems by breeding plant resistance, identifying metabolic targets for insecticide development, etc.

  1. Efeito de diferentes componentes no meio de cultura na produção de alcalóides em tecidos de calos de Cereus peruvianus (Cactaceae - DOI: 10.4025/actascibiolsci.v27i1.1355 Effect of different culture medium components on production of alkaloid in callus tissues of Cereus peruvianus (Cactaceae - DOI: 10.4025/actascibiolsci.v27i1.1355

    Directory of Open Access Journals (Sweden)

    Claudete Aparecida Mangolin

    2005-03-01

    Full Text Available Tecidos de calos em cultura prolongada de Cereus peruvianus foram utilizados para investigar a produção de alcalóides pelos calos quando mantidos em meio de cultura suplementados com diferentes concentrações de tirosina, com diferentes níveis de 2,4-D/cinetina, e contendo NaCl. Foi analisado também, o padrão de isozimas α- e β-esterases, para investigar a expressão diferencial de genes nos calos subcultivados com as diferentes concentrações de tirosina. A maior quantidade de alcalóides foi obtida a partir de calos mantidos em meio contendo 200 mg/L de tirosina. A adição de tirosina no meio induziu uma expressão gênica diferencial para a síntese de isozimas α- e β-esterases. Duas α-carboxilesterases (EC 3.1.1.1 e uma α/β-arilesterase (EC 3.1.1.2 foram induzidas nos calos, uma α/β-acetilesterase (EC 3.1.1.6 e uma α-carboxilesterase foram detectadas como bandas mais intensamente coradas, enquanto duas α/β-carboxilesterases não foram detectadas ou foram fracamente coradas após a adição de tirosina como precursor no meio de culturaThe production of alkaloids from Cereus peruvianus callus cultured in medium supplemented with different tyrosine concentrations, different 2,4-D/kinetin levels, and with NaCl was investigated using long-term callus culture. α- and β-Esterase isozyme patterns were also analyzed to investigate the differential gene expression in callus subcultured with the different tyrosine concentrations. The greatest amounts of alkaloids were obtained from callus maintained in medium containing 200 mg/L of tyrosine. Tyrosine induced a differential gene expression for the synthesis of specific α- and β -esterase isozymes. Two α-carboxylesterases (EC 3.1.1.1 and one α/β-arylesterase (EC 3.1.1.2 were induced in callus, one α/β-acetylesterase (EC 3.1.1.6 and two α-carboxylesterase were detected as more intensely stained while two α/β-carboxylesterases were absent or detected as more weakly stained

  2. Coccolithophores: Functional Biodiversity, Enzymes and Bioprospecting

    Directory of Open Access Journals (Sweden)

    Michael J. Allen

    2011-04-01

    Full Text Available Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an ‘in house‘ enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.

  3. Coccolithophores: functional biodiversity, enzymes and bioprospecting.

    Science.gov (United States)

    Reid, Emma L; Worthy, Charlotte A; Probert, Ian; Ali, Sohail T; Love, John; Napier, Johnathan; Littlechild, Jenny A; Somerfield, Paul J; Allen, Michael J

    2011-01-01

    Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an 'in house' enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.

  4. Clopidogrel Bioactivation and Risk of Bleeding in Patients Cotreated With Angiotensin-Converting Enzyme Inhibitors After Myocardial Infarction

    DEFF Research Database (Denmark)

    Kristensen, Karl E.; Zhu, Hao-Jie; Wang, Xinwen

    2014-01-01

    Clopidogrel is an oral antiplatelet prodrug, the majority of which is hydrolyzed to an inactive metabolite by hepatic carboxylesterase 1 (CES1). Most angiotensin-converting enzyme inhibitors (ACEIs) are also metabolized by this enzyme. We examined the effects of ACEIs on clopidogrel bioactivation...... in vitro and linked the results with a pharmacoepidemiological study. In vitro, ACEIs inhibited CES1-mediated hydrolysis of a model substrate, and trandolapril and enalapril increased formation of clopidogrel active metabolite. In 70,934 patients with myocardial infarction, hazard ratios for clinically...... significant bleeding in ACEI-treated patients cotreated with or without clopidogrel were 1.10 (95% confidence interval (CI): 0.97-1.25, P = 0.124) and 0.90 (95% CI: 0.81-0.99, P = 0.025), respectively, as compared with patients who did not receive ACEIs. This difference was statistically significant (P = 0...

  5. Ecological Fitness of Non-vector Planthopper Sogatella furcifera on Rice Plants Infected with Rice Black Streaked Dwarf Virus

    Institute of Scientific and Technical Information of China (English)

    HE Xiao-chan; XU Hong-xing; ZHENG Xu-song; YANG Ya-jun; GAO Guang-chun; PAN Jian-hong; LU Zhong-xian

    2012-01-01

    We evaluated the effects of rice black streak dwarf virus (RBSDV)-infested rice plants on the ecological parameters and its relevant defensive and detoxification enzymes of white-backed planthopper (WBPH) in laboratory for exploring the relationship between RBSDV and the non-vector planthopper.The results showed that nymph survival rate,female adult weight and fecundity,and egg hatchability of WBPH fed on RBSDV-infested rice plants did not markedly differ from those on healthy plants,whereas the female adult longevity and egg duration significantly shortened on diseased plants.Furthermore,significantly higher activities of defensive enzymes (dismutase,catalase and peroxidase) and detoxification enzymes (acetylcholinesterase,carboxylesterase and glutathione S-transferase) were found in WBPH adults fed on infected plants.Results implied that infestation by RBSDV increased the ecological fitness of non-vector planlhopper population.

  6. Acaricidal activity of Ocimum basilicum and Spilanthes acmella against the ectoparasitic tick, Rhipicephalus (Boophilus) microplus (Arachinida: Ixodidae).

    Science.gov (United States)

    Veeramani, V; Sakthivelkumar, S; Tamilarasan, K; Aisha, S O; Janarthanan, S

    2014-09-01

    The ectoparasitic tick, Rhipicephalus (Boophilus) microplus collected at various cattle farms in and around Chennai was subjected to treatment of different crude solvent extracts of leaves of Ocimum basilicum and Spilanthes acmella for acaricidal activity. Among various solvent extracts of leaves of O. basilicum and S. acmella used, chloroform extract of O. basilicum at concentrations between 6% and 10% exhibited 70% and 100% mortality of ticks when compared to control. The LC50 and LC90 values of the chloroform extract of leaves of O. basilicum treatment on the ticks after 24 h were observed as 5.46% and 7.69%. Quantitative and qualitative analysis of α- and β- carboxylesterase enzymes in the whole gut homogenate of cattle tick, R. microplus treated with chloroform extract of leaves of O. basilicum revealed higher level of activities for the enzymes. This indicated that there was an induced response in the tick, R. microplus against the toxic effects of the extract of O. basilicum.

  7. Toxic interaction of tetraisopropylpyrophosphoramide and propoxur: some insights into the mechanisms.

    Science.gov (United States)

    Gupta, R C; Kadel, W L

    1990-01-01

    Propoxur with a non-toxic dose (5 mg/kg) administered intraperitoneally (ip) in tetraisopropylpyrophosphoramide (iso-OMPA, 1 mg/kg) pretreated rats subcutaneously, sc) produced severe intoxication of anticholinesterase nature. The observed severity was comparable to that caused by an acute sublethal dose of propoxur (15 mg/kg) suggesting at least threefold potentiation of toxicity. Either drug given alone produced neither signs of toxicity nor alterations in acetylcholinesterase (AChE) activity, while carboxylesterase (CarbE) activity was markedly reduced indicating tremendous nonspecific binding. The administration of iso-OMPA followed by propoxur elicited inhibition of AChE to a critical level and produced severe intoxication. These results suggested that iso-OMPA induced potentiation of propoxur toxicity stemmed through irreversible inhibition of CarbE.

  8. Preparation of cobalt nanoparticles from polymorphic bacterial templates: A novel platform for biocatalysis.

    Science.gov (United States)

    Jang, Eunjin; Shim, Hyun-Woo; Ryu, Bum Han; An, Deu Rae; Yoo, Wan Ki; Kim, Kyeong Kyu; Kim, Dong-Wan; Kim, T Doohun

    2015-11-01

    Nanoparticles have gathered significant research attention as materials for enzyme immobilization due to their advantageous properties such as low diffusion rates, ease of manipulation, and large surface areas. Here, polymorphic cobalt nanoparticles of varied sizes and shapes were prepared using Micrococcus lylae, Bacillus subtilis, Escherichia coli, Paracoccus sp., and Haloarcula vallismortis as bacterial templates. Furthermore, nine lipases/carboxylesterases were successfully immobilized on these cobalt nanoparticles. Especially, immobilized forms of Est-Y29, LmH, and Sm23 were characterized in more detail for potential industrial applications. Immobilization of enzymes onto cobalt oxide nanoparticles prepared from polymorphic bacterial templates may have potential for efficient hydrolysis on an industrial-scale, with several advantages such as high retention of enzymatic activity, increased stability, and strong reusability.

  9. Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes.

    Science.gov (United States)

    Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang

    2014-01-01

    Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented.

  10. [Therapeutic drug monitoring of rufinamide].

    Science.gov (United States)

    Bentué-Ferrer, Danièle; Tribut, Olivier; Verdier, Marie-Clémence

    2012-01-01

    Rufinamide is a third-generation antiepileptic drug, available since early 2010 in France. It is indicated in combination therapy in the Lennox-Gastaut syndrome from the age of 4. It has orphan drug status. The bioavailability of rufinamide is high, but decreases with the dose and increases with food intake. Rufinamide is not metabolized by cytochromes but hydrolyzed by a carboxylesterase in an inactive carboxylic derivative. Elimination is mainly renal. The half-life varies from 6 to 10h. Although established from relatively few studies, exposure efficacy and exposure toxicity relationships are argued. A plasma concentration of 15 mg/L, obtained with a standard regimen, reduces the number of seizures of 25%. Few factors of intrinsic variability are described. There are few clinically significant pharmacokinetic interactions and they concern combinations with other antiepileptic drugs, especially valproate. Although there is no validated therapeutic range, the level of evidence for this therapeutic drug monitoring has been estimated at "possibly useful".

  11. Aquatic ecotoxicity of a pheromonal antagonist in Daphnia magna and Desmodesmus subspicatus.

    Science.gov (United States)

    Rosa, Esmeralda; Barata, Carlos; Damásio, Joana; Bosch, Maria Pilar; Guerrero, Angel

    2006-09-12

    Evaluation of the ecotoxicological potential of (Z)-11-hexadecenyl trifluoromethyl ketone (Z11-16:TFMK), a new biorational agent with high prospective features to control the Mediterranean corn borer Sesamia nonagrioides in infested maize fields, in comparison to the parent pheromone compound (Z)-11-hexadecenyl acetate (Z11-16:Ac) is described. Acute and sublethal toxicity tests of both compounds against the cladoceran Daphnia magna and the chlorophyte Desmodesmus subspicatus were conducted, the endpoints being immobilisation and feeding inhibition for Daphnia and growth rate inhibition for Desmodesmus. In addition, effects on B esterases including cholinesterase and carboxylesterase activities in Daphnia were also assessed to evaluate the mode of action of both chemicals. Toxicities of both compounds were moderate with EC(50) values ranging from 3.11 to 103.74mgl(-1) in algae growth, from 0.07 to 1.20mgl(-1) in Daphnia survival, and from 0.10 to 0.53mgl(-1) in Daphnia feeding rate. In all cases Z11-16:TFMK was more toxic than the naturally occurring pheromone component. Serine esterase assays showed a strong inhibition of the carboxylesterase activities in Daphnia at concentrations with apparently no effects on survival or feeding, suggesting that inhibition of other key esterases may be the possible mechanism of toxicity of this compound. The results obtained have been related with some physico-chemical properties of the compounds, such as water solubility and octanol-water partition coefficient, suggesting that Z11-16:TFMK may affect aquatic organisms at lower concentrations than expected from non-polar narcosis.

  12. Incongruent nuclear and mitochondrial genetic structure of new world screwworm fly populations due to positive selection of mutations associated with dimethyl- and diethyl-organophosphates resistance.

    Science.gov (United States)

    Bergamo, Luana Walravens; Fresia, Pablo; Azeredo-Espin, Ana Maria L

    2015-01-01

    Livestock production is an important economic activity in Brazil, which has been suffering significant losses due to the impact of parasites. The New World screwworm (NWS) fly, Cochliomyia hominivorax, is an ectoparasite and one of the most important myiasis-causing flies endemic to the Americas. The geographic distribution of NWS has been reduced after the implementation of the Sterile Insect Technique (SIT), being eradicated in North America and part of Central America. In South America, C. hominivorax is controlled by chemical insecticides, although indiscriminate use can cause selection of resistant individuals. Previous studies have associated the Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 to resistance of diethyl and dimethyl-organophosphates insecticides, respectively. Here, we have sequenced a fragment of the carboxylesterase E3 gene (ChαE7), comprising part of intron iII, exon eIII, intron iIII and part of exon eIV, and three mitochondrial gene sequences (CR, COI and COII), of NWS flies from 21 locations in South America. These markers were used for population structure analyses and the ChαE7 gene was also investigated to gain insight into the selective pressures that have shaped its evolution. Analysis of molecular variance (AMOVA) and pairwise FST analysis indicated an increased genetic structure between locations in the ChαE7 compared to the concatenated mitochondrial genes. Discriminant analysis of principal components (DAPC) and spatial analysis of molecular variance (SAMOVA) indicated different degrees of genetic structure for all markers, in agreement with the AMOVA results, but with low correlation to geographic data. The NWS fly is considered a panmitic species based on mitochondrial data, while it is structured into three groups considering the ChαE7 gene. A negative association between the two mutations related to organophosphate resistance and Fay & Wu's H significant negative values for the exons, suggest

  13. In vitro metabolism and drug-drug interaction potential of UTL-5g, a novel chemo- and radioprotective agent.

    Science.gov (United States)

    Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick; Li, Jing

    2014-12-01

    N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values 5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g.

  14. Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues.

    Science.gov (United States)

    Ozaki, Hitomi; Sugihara, Kazumi; Watanabe, Yoko; Fujino, Chieri; Uramaru, Naoto; Sone, Tomomichi; Ohta, Shigeru; Kitamura, Shigeyuki

    2013-12-01

    Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure-metabolic activity relationship was examined. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.

  15. Hydrolytic metabolism of phenyl and benzyl salicylates, fragrances and flavoring agents in foods, by microsomes of rat and human tissues.

    Science.gov (United States)

    Ozaki, Hitomi; Sugihara, Kazumi; Tamura, Yuki; Fujino, Chieri; Watanabe, Yoko; Uramaru, Naoto; Sone, Tomomichi; Ohta, Shigeru; Kitamura, Shigeyuki

    2015-12-01

    Salicylates are used as fragrance and flavor ingredients for foods, as UV absorbers and as medicines. Here, we examined the hydrolytic metabolism of phenyl and benzyl salicylates by various tissue microsomes and plasma of rats, and by human liver and small-intestinal microsomes. Both salicylates were readily hydrolyzed by tissue microsomes, predominantly in small intestine, followed by liver, although phenyl salicylate was much more rapidly hydrolyzed than benzyl salicylate. The liver and small-intestinal microsomal hydrolase activities were completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Phenyl salicylate-hydrolyzing activity was co-eluted with carboxylesterase activity by anion exchange column chromatography of the Triton X-100 extracts of liver and small-intestinal microsomes. Expression of rat liver and small-intestinal isoforms of carboxylesterase, Ces1e and Ces2c (AB010632), in COS cells resulted in significant phenyl salicylate-hydrolyzing activities with the same specific activities as those of liver and small-intestinal microsomes, respectively. Human small-intestinal microsomes also exhibited higher hydrolyzing activity than liver microsomes towards these salicylates. Human CES1 and CES2 isozymes expressed in COS cells both readily hydrolyzed phenyl salicylate, but the activity of CES2 was higher than that of CES1. These results indicate that significant amounts of salicylic acid might be formed by microsomal hydrolysis of phenyl and benzyl salicylates in vivo. The possible pharmacological and toxicological effects of salicylic acid released from salicylates present in commercial products should be considered.

  16. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

    Science.gov (United States)

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-05-07

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

  17. Simvastatin requires activation in accessory cells to modulate T-cell responses in asthma and COPD.

    Science.gov (United States)

    Knobloch, Jürgen; Yakin, Yakup; Körber, Sandra; Grensemann, Barbara; Bendella, Zeynep; Boyaci, Niyazi; Gallert, Willem-Jakob; Yanik, Sarah Derya; Jungck, David; Koch, Andrea

    2016-10-05

    T-cell-dependent airway and systemic inflammation triggers the progression of chronic obstructive pulmonary disease (COPD) and asthma. Retrospective studies suggest that simvastatin has anti-inflammatory effects in both diseases but it is unclear, which cell types are targeted. We hypothesized that simvastatin modulates T-cell activity. Circulating CD4+ and CD8+ T-cells, either pure, co-cultured with monocytes or alveolar macrophages (AM) or in peripheral blood mononuclear cells (PBMCs), were ex vivo activated towards Th1/Tc1 or Th2/Tc2 and incubated with simvastatin. Markers for Th1/Tc1 (IFNγ) and Th2/Tc2 (IL-5, IL-13) were measured by ELISA; with PBMCs this was done comparative between 11 healthy never-smokers, 11 current smokers without airflow limitation, 14 smokers with COPD and 11 never-smokers with atopic asthma. T-cell activation induced IFNγ, IL-5 and IL-13 in the presence and absence of accessory cells. Simvastatin did not modulate cytokine expression in pure T-cell fractions. β-hydroxy-simvastatin acid (activated simvastatin) suppressed IL-5 and IL-13 in pure Th2- and Tc2-cells. Simvastatin suppressed IL-5 and IL-13 in Th2-cells co-cultivated with monocytes or AM, which was partially reversed by the carboxylesterase inhibitor benzil. Simvastatin suppressed IL-5 production of Th2/Tc2-cells in PBMCs without differences between cohorts and IL-13 stronger in never-smokers and asthma compared to COPD. Simvastatin induced IFNγ in Th1/Tc1-cells in PBMCs of all cohorts except asthmatics. Simvastatin requires activation in accessory cells likely by carboxylesterase to suppress IL-5 and IL-13 in Th2/Tc2-cells. The effects on Il-13 are partially reduced in COPD. Asthma pathogenesis prevents simvastatin-induced IFNγ up-regulation. Simvastatin has anti-inflammatory effects that could be of interest for asthma therapy.

  18. Highly selective biomarkers for pesticides developed in Eisenia fetida using SELDI-TOF MS.

    Science.gov (United States)

    Park, Doo-San; Jeon, Hwang-Ju; Park, Eun-Sil; Bae, In Kyung; Kim, Yong-Eun; Lee, Sung-Eun

    2015-03-01

    The repeated use of pesticides, and their subsequent residues, has contributed to severe adverse effects on the environment, including risks to human health. Therefore, it is important to assess the quality of the environment to ensure it remains free from pesticide residues. The six pesticides tested in this study showed high mortality on Eisenia fetida with LC50 values ranging from 7.7 to 37.9 g L(-1). The strongest lethal effect resulted from the organochlorine insecticide endosulfan (LC50=7.7 g L(-1)). Following exposure to the carbamate pesticides, acetylcholinesterase activity in E. fetida decreased dramatically in comparison to the control. Carboxylesterase activity was only lowered in E. fetida exposed to propoxur, when compared to the control. The remaining five pesticides had no significant effect on carboxylesterase activity in E. fetida. In order to discover pesticide-specific biomarkers with differentially expressed proteins after exposure to pesticides, protein patterns of pesticide-treated E. fetida were analyzed using SELDI-TOF MS with Q10 ProteinChips. Protein patterns were compared with their intensities at the same mass-to-charge ratios (m/z). All 42 peaks had intensities with associated p-values less than 0.089, and 40 of these peaks had associated p-values of 0.05. Using SELDI-TOF MS technology, selective biomarkers for the six pesticides tested were found in E. fetida; four proteins with 5425, 5697, 9523, and 9868 m/z were consistently observed in the earthworms following exposure to the carbamates.

  19. 转Bt基因南林895杨对杨扇舟蛾体内酶的影响%Effects of Bt Nanlin 895 Poplar on the Metabolic Enzymes of Scarce Chocolate Tip, Clostera anachoreta

    Institute of Scientific and Technical Information of China (English)

    张雁; 郭同斌; 诸葛强

    2012-01-01

    The larvae of scarce chocolate tip, Clostera anachoreta, were fed with leaves of Bt Nanlin 895 poplar to study effects of Bt protein on the metabolic enzymes of the pest. The results showed that the activities of eBterase and carboxylestera.se were inhibited by Bt protein. The esterase activity of the larvae reduced after being fed with the transgenic poplar leaves in 4 - 48 h and the activity of carboxylesterase rose during the initial 4 h but declined after then. The activity of multi-function oxidase in the larvae slightly declined after being fed with the transgenic leaves in 24 h, but the difference was not significant from CK. The activity of glutathione-S-transferase did not significantly change during the initial 36 h, but was inhibited by the Bt protein in 48 h. The activity of acetylcholinesterase was not affected by the feeding. The activity of superoxide dismutase rose in 12 and then declined during 12 -48 h. The activity of peroxidase rose during 4 - 48 h while the activity of catalase declined during 4 - 12 h and rose from 24-48 h. Therefore, the transgenic poplar inhibited the activities of esterase and carboxylesterase, disturbed the protect enzyme system and had an insecticidal effect of the larvae.%以转Bt基因南林895杨树叶片饲喂杨扇舟蛾,研究Bt毒蛋白对害虫体内代谢酶的影响.结果表明:酯酶和羧酸酯酶明显受到抑制.中肠酯酶活性在取食4 ~48 h内持续下降,羧酸酯酶活性在前期4h有所上升但随后持续下降.多功能氧化酶活性从24h起,受抑而下降,但不显著.谷胱甘肽S-转移酶活性在前36 h内波动不大,48 h后显著受抑.乙酰胆碱酶的活性无明显受抑.超氧化物歧化酶活性12 h后略升高,然而在取食12~48 h活性持续下降,与之相反,过氧化物酶活性在4~48 h呈上升状态.过氧化氢酶活性在4~12h呈下降状态,24 ~48h呈上升状态.转Bt基因杨树主要通过抑制酯酶和羧酸酯酶并扰乱超氧化物歧化酶、

  20. Effects of dexamethasone coadministered with oseltamivir on the pharmacokinetics of oseltamivir in healthy volunteers

    Directory of Open Access Journals (Sweden)

    Jang K

    2017-03-01

    Full Text Available Kyungho Jang,1,2,* Min-Kyoung Kim,3,4,* Jaeseong Oh,1 SeungHwan Lee,1 Joo-Youn Cho,1 Kyung-Sang Yu,1 Tai Kiu Choi,3 Sang-Hyuk Lee,3,4 Kyoung Soo Lim4 1Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, Seoul, 2Center for Clinical Pharmacology and Biomedical Research Institute, Chonbuk National University Medical School, Jeonju, 3Department of Psychiatry, 4Department of Clinical Pharmacology and Therapeutics, CHA University School of Medicine and CHA Bundang Medical Center, Seongnam, Republic of Korea *These authors contributed equally to this work Purpose: Oseltamivir is widely used in the treatment and prophylaxis of influenza A and B viral infections. It is ingested as an oral prodrug that is rapidly metabolized by carboxylesterase 1 (CES1 to its active form, oseltamivir carboxylate. Dexamethasone is also used in the treatment of acute respiratory distress syndrome, a severe complication of influenza; however, its influence on the pharmacokinetics (PK of oseltamivir is controversial. The aim of this study was to investigate the effects of coadministering oseltamivir and dexamethasone on the PK of oseltamivir in healthy volunteers. Methods: An open-label, two-period, one-sequence, multiple-dose study was conducted in 19 healthy male volunteers. Oseltamivir (75 mg was orally administered on Day 1 and Day 8, and dexamethasone (1.5 mg was administered once daily from Day 3 to Day 8. Serial blood and urine samples were collected for PK analysis of oseltamivir and oseltamivir carboxylate on Day 1 and Day 8. Oseltamivir and oseltamivir carboxylate concentrations in plasma and urine were determined using liquid chromatography–tandem mass spectrometry. Results: Area under the plasma concentration–time curve (AUC of oseltamivir and oseltamivir carboxylate decreased after dexamethasone treatment for 6 days. The geometric mean ratio (90% confidence interval of the metabolic ratio

  1. 杀虫剂处理对水稻负泥虫体内解毒酶的影响%Effects of Pesticides Treatment on the Activity of Detoxification Enzymes in the Larvae of O. oryzae

    Institute of Scientific and Technical Information of China (English)

    于磊; 王丽艳; 张海燕

    2011-01-01

    [目的]研究不同杀虫剂对水稻负泥虫(Oulema onzae)体内解毒酶的影响.[方法]以水稻负泥虫为研究对象,分析幼虫在高效氯氰菊酯、阿维菌素和吡虫啉3种杀虫剂处理1、4、9和15 d后,幼虫体内羧酸酯酶及谷胱甘肽转移酶活性变化.[结果]随着取食时间的延长,3种药剂处理的幼虫体内羧酸酯酶活性均较对照增高,且差异明显;而3种药剂处理的谷胱甘肤转移酶活性均低于对照,但差异不明显.[结论]解毒酶的变化在害虫抵抗杀虫剂处理过程中起到指示作用,该研究可为水稻负泥虫抗药性研究奠定基础.%[ Objective ] The aim was to study the effects of different pesticides on the activity of detoxification enzymes in the larvae of O.oryzae. [ Method ] As the model of the larvae of O. oryzae, the change of carboxylesterase and glutathione transferase in larvaes fed with rice leaves treated 1, 4, 9 and 15 days by 3 insecticides, beta-cypermethrin, abamectin and imidacloprid, were analyzed. [ Result ] The results showed that carboxylesterase activity of 3 insecticides treatments in the larvae was higher than the control with the time increased, and the difference among 3 insecticides treatments was significant. While glutathione transferase activity of 3 insecticides treatments in the larvae was lower than the control with the time increased, but the difference among 3 insecticides treatments was no significant. [ Conclusion ] Detoxification enzymes can acted as markers in the process of insecticide pest control. The study can lay a basis for researching of the resistance mechanisms to insecticides of O. oryzae.

  2. Effects of sublethal doses of phoxim on enzyme activity of Pealius mori (Takahashi)%辛硫磷亚致死剂量对桑粉虱解毒酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    柴建萍; 倪婧; 江秀均; 罗雁婕; 谢道燕; 达爱斯; 黄平

    2013-01-01

    [目的]研究辛硫磷对桑粉虱成虫的毒力及其亚致死剂量作用下桑粉虱成虫谷胱甘肽S-转移酶(GST)、羧酸酯酶(CarE)、乙酰胆碱酯酶(AchE)的活性变化,为桑粉虱抗药性研究及杀虫剂合理使用提供依据.[方法]采用闪烁管药膜法测定辛硫磷对桑粉虱亚致死剂量LC10、LC30、LC50,玻璃均浆器冰浴提取经过处理的桑粉虱酶液,用BIO-RAD全波长微孔板酶标读数仪测定GST、CarE、AchE.[结果]辛硫磷对桑粉虱成虫亚致死剂量LC10、LC30、LC50分别为0.0090、0.0899、0.4427 μg/mL,随着辛硫磷亚致死剂量浓度增大,桑粉虱成虫GST活性显著增大、CarE及AchE活性显著下降.[结论]GST在桑粉虱对辛硫磷的解毒代谢中发挥作用.%[Obejective]The virulence and effects of sublethal dose of phoixm on activities of glutathione s-tranferase,car-boxylesterase and acetylcholinesterase from pest insect Pealius mori (Takahashi) were studied to provide references for P.mori (Takahashi) resistance research and suitable application of pesticide.[Method]The sublethal doses of phoxim LC10,LC30,LC50 were measured by glass tube residual film.The enzyme solution of P.mori(Takahashi) was extracted by glass BioMasher.The enzyme activities of glutathione s-tranferase,carboxylesterase and acetylcholinesterase of P.mori (Taka-hashi) was detected by BIO-RAD Multiskan Spectrum.[Result]The enzyme activity of glutathione s-tranferase increased notably as the sublethal doses of phoxim disoposal concentration went up,while the enzyme activities of carboxylesterase and acetylcholinesterase obviously decreased.[Conclusion]The glutathione s-tranferase played a role in the course of phoixm detoxifcation in P.mori (Takahashi).

  3. Effects of alpha-mangostin from mangosteen pericarp extract and imidacloprid on Nilaparvata lugens (Stal.) and non-target organisms: toxicity and detoxification mechanism.

    Science.gov (United States)

    Bullangpoti, Vasakorn; Visetson, Suraphon; Milne, John; Milne, Manthana; Sudthongkong, Chaiwud; Pronbanlualap, Somchai

    2007-01-01

    The brown planthopper, Nilaparvato lugens Stat. (BPH) is the most devastating insect pest in rice fields. Outbreaks of BPH, which are resistant to many synthetic insecticides, can cause total rice crop loss. This research was done to evaluate the efficiency of extracts of mangosteen pericarp (Garcina mangostana L.) as an alternative control of BPH Thailand strain. Topical spraying was applied to various stages of nymphal and adult BPH to determine toxicity. An ethanol extract of mangosteen pericarp extract gave the best control of BPH, with LC50 of 4.5% w/v (r2 = 0.95) with 3rd instar BPH nymphs when compared with the other solvents, hexane, acetone and dichloromethane. The active compound, alpha-mangostin showed an LC50 of 5.44%w/v (r2 = 0.88). The toxicity of this extract was less than that of Imidacloprid which showed an LC50 of 0.0042% w/v (r2 = 0.99). The toxicity to non-target organisms was determined. This extract showed toxicity to guppies ((LC50 = 2.53 and 4.27 ppm for females and males, respectively; r2 = 0.97 and 0.97, respectively), bees (LC50 = 4.38% w/v, r2 = 0.95) and mice (no oral acute toxicity and no dermal inflammation but showed eye irritation in 1 day which became normal within 3 days). In vitro detoxification enzyme activities of carboxylesterase, acetylcholinesterase and glutathione-s-transferase from BPH after 24 hours exposure were also observed. Carboxylesterase showed stronger activity than other enzymes. Toxicity in terms of LC50 values of both the extract and imidacloprid treatments increased in each generation. The LC50 values for each generation were 4.22-6.67 after sequential spraying. After the ethanol extract was kept at 4 degrees C, room temperature and 55 degrees C for 3 months, the quantity of alpha-mangostin and the BPH control efficiency was lower at 55 degrees C than those for other temperatures. The results from this research indicate that mangosteen pericarp extract can be an alternative insecticide for the control of BPH

  4. Pyrethroid Resistance in Malaysian Populations of Dengue Vector Aedes aegypti Is Mediated by CYP9 Family of Cytochrome P450 Genes

    Science.gov (United States)

    Ishak, Intan H.; Kamgang, Basile; Ibrahim, Sulaiman S.; Riveron, Jacob M.; Irving, Helen

    2017-01-01

    Background Dengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance. Methodology/Principal Findings Genome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise

  5. Effects of Mitragynine and a Crude Alkaloid Extract Derived from Mitragyna speciosa Korth. on Permethrin Elimination in Rats

    Directory of Open Access Journals (Sweden)

    Kachamas Srichana

    2015-03-01

    Full Text Available Detoxification and elimination of permethrin (PM are mediated by hydrolysis via carboxylesterase (CES. Mitragyna speciosa (kratom contains mitragynine (MG and other bioactive alkaloids. Since PM and MG have the same catalytic site and M. speciosa is usually abused by adding other ingredients such as pyrethroid insecticides, the effects of MG and an alkaloid extract (AE on the elimination of PM were investigated in rats. Rats were subjected to single and multiple pretreatment with MG and AE prior to receiving a single oral dose (460 mg/kg of PM. Plasma concentrations of trans-PM and its metabolite phenoxybenzylalcohol (PBAlc were measured. The elimination rate constant (kel and the elimination half-life (t1/2 el of PM were determined, as well as the metabolic ratio (PMR. A single and multiple oral pretreatment with MG and AE altered the plasma concentration-time courses of both trans-PM and PBAlc during 8–22 h, decreased the PMRs, delayed elimination of PM, but enhanced elimination of PBAlc. Results indicated that PM–MG or AE toxicokinetic interactions might have resulted from the MG and AE interfering with PM hydrolysis. The results obtained in rats suggest that in humans using kratom cocktails containing PM, there might be an increased risk of PM toxicity due to inhibition of PM metabolism and elimination.

  6. Toxicity of Diphenyl-pentenone Against Three Aphids(Homoptera:Aphididae) and Its Effects on the Esterases and Glutathione S-transferase

    Institute of Scientific and Technical Information of China (English)

    Gao Ping(高平); Gan Mingzhe; Liu Shigui

    2004-01-01

    The most provocative aspect of this study is its original findings on the toxicity of diphenyl-pentenone(1,5-diphenyl-2-penten-1-one, DP) against the three kinds of aphids (Macrosiphum granarium Kirby, Lipaphis erysimi(kaltenbach), Schizaphis aurantiae(Fosc.)and the inhibitory effects of DP against detoxification enzyme system of the aphid (S aurantiae). The result of bioassay in the laboratory shows that the product has strong contact activity and very good antifeeding activity, with higher efficacy than anabasine and nicotine, two botanical aphidicides. The median lethal concentrations of the three products against the apterous adult (M granarium) are 175.85, 217.23 and 245.22 mg/L, respectively, at 24 h of pest treatment. DP is inferior to methomyl in contact assay but superior in antifeeding activity assay against the three aphides. DP has strong inhibitory effects on nonspecific esterases, carboxylesterase of aphid (S aurantiae), but it can not inhibit AchE. DP also has strong inhibitory effects on glutathione S-transferase of the aphid.

  7. The role of detoxifying enzymes in the resistance of the cowpea aphid (Aphis craccivora Koch to thiamethoxam

    Directory of Open Access Journals (Sweden)

    Abdallah Ibrahim Saleh

    2016-01-01

    Full Text Available The cowpea aphid (Aphis craccivora Koch is considered a serious insect pest attacking several crops. We carried out biochemical studies to elucidate the role of the metabolising enzymes in conferring resistance to thiamethoxam, in two strains (resistant and susceptible of the cowpea aphid. Bioassay experiments showed that the thiamethoxam selected strain developed a 48 fold resistance after consecutive selection with thiamethoxam for 12 generations. This resistant strain also exhibited cross-resistance to the tested carbamates; pirimicarb and carbosulfan, organophosphorus (malathion, fenitrothion, and chlorpyrifos-methyl, and the neonicotinoid (acetamiprid. Synergism studies have indicated that S,S,S-tributyl phosphorotrithioate (DEF, a known inhibitor for esterases, increased thiamethoxam toxicity 5.58 times in the resistant strain compared with the susceptible strain. Moreover, the biochemical determination revealed that carboxylestersae activity was 30 times greater in the resistant strain than in the susceptible strain. In addition, the enzyme activity of glutathione S-transferase (GST and mixed function oxidases (mfo increased only in the resistant strain 3.7 and 2.7 times, respectively, in relation to the susceptible (the control. Generally, our results suggest that the higher activity of the detoxifying enzymes, particularly carboxylesterase, in the resistant strain of the cowpea aphid, apparently have a significant role in endowing resistance to thiamethoxam, although additional mechanisms may contribute.

  8. Evaluation of released malathion and spinosad from chitosan/alginate/gelatin capsules against Culex pipiens larvae

    Directory of Open Access Journals (Sweden)

    Badawy MEI

    2016-09-01

    Full Text Available Mohamed EI Badawy,1 Nehad EM Taktak,2 Osama M Awad,2 Souraya A Elfiki,2 Nadia E Abou El-Ela2 1Department of Pesticide Chemistry and Technology, Faculty of Agriculture, 2Department of Tropical Health, High Institute of Public Health, Alexandria University, Alexandria, Egypt Abstract: Efficacy of spinosad and malathion loaded in eco-friendly biodegradable formulations was evaluated for controlling Culex pipiens larvae. Malathion (organophosphorus larvicide and spinosad (naturally derived insecticide were loaded on chitosan/alginate/gelatin capsules. Capsules were characterized by size measurement, scanning electron microscopy, Fourier transform infrared spectroscopy, and water uptake. In vitro release kinetics of the larvicides was studied in the running and stagnant water. Biochemical studies on the larvae treated with technical and formulated insecticides were also demonstrated. The results indicated that the released spinosad was active for a long time up to 48 and 211 days in the running and stagnant water, respectively. However, the capsules loaded with malathion showed larvicidal activity for 20 and 27 days in the running and stagnant water, respectively. Technical and formulated malathion and spinosad had an inhibition effect on acetylcholinesterase, carboxylesterase, and glutathione S-transferase. The results proved that the prepared capsules consisting of biodegradable polymers containing larvicides could be effective as controlled-release formulation against C. pipiens larvae for a long period. Keywords: chitosan capsules, larvicide, controlled-release formulation, swelling, mosquitocidal activity, Culex pipiens, biochemical study

  9. De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube

    Directory of Open Access Journals (Sweden)

    Domenico Iaria

    2016-01-01

    Full Text Available In olive (Olea europaea L., the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cultivars, have a distinct gene expression profile and many of the differentially expressed sequences between the samples fall within gene families involved in the development of the pollen tube, such as lipase, carboxylesterase, pectinesterase, pectin methylesterase, and callose synthase. Moreover, different genes involved in signal transduction, transcription, and growth are overrepresented. The analysis also allowed us to identify members in actin and actin depolymerization factor and fibrin gene family and member of the Ca2+ binding gene family related to the development and polarization of pollen apical tip. The whole transcriptomic analysis, through the identification of the differentially expressed transcripts set and an extended functional annotation analysis, will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth in the olive.

  10. De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube.

    Science.gov (United States)

    Iaria, Domenico; Chiappetta, Adriana; Muzzalupo, Innocenzo

    2016-01-01

    In olive (Olea europaea L.), the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cultivars, have a distinct gene expression profile and many of the differentially expressed sequences between the samples fall within gene families involved in the development of the pollen tube, such as lipase, carboxylesterase, pectinesterase, pectin methylesterase, and callose synthase. Moreover, different genes involved in signal transduction, transcription, and growth are overrepresented. The analysis also allowed us to identify members in actin and actin depolymerization factor and fibrin gene family and member of the Ca(2+) binding gene family related to the development and polarization of pollen apical tip. The whole transcriptomic analysis, through the identification of the differentially expressed transcripts set and an extended functional annotation analysis, will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth in the olive.

  11. Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

    Science.gov (United States)

    Mangas, I; Vilanova, E; Benabent, M; Estévez, J

    2014-02-10

    Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins.

  12. Hydrolytic Fate of 3/15-Acetyldeoxynivalenol in Humans: Specific Deacetylation by the Small Intestine and Liver Revealed Using in Vitro and ex Vivo Approaches

    Science.gov (United States)

    Ajandouz, El Hassan; Berdah, Stéphane; Moutardier, Vincent; Bege, Thierry; Birnbaum, David Jérémie; Perrier, Josette; Di Pasquale, Eric; Maresca, Marc

    2016-01-01

    In addition to deoxynivalenol (DON), acetylated derivatives, i.e., 3-acetyl and 15-acetyldexynivalenol (or 3/15ADON), are present in cereals leading to exposure to these mycotoxins. Animal and human studies suggest that 3/15ADON are converted into DON after their ingestion through hydrolysis of the acetyl moiety, the site(s) of such deacetylation being still uncharacterized. We used in vitro and ex vivo approaches to study the deacetylation of 3/15ADON by enzymes and cells/tissues present on their way from the food matrix to the blood in humans. We found that luminal deacetylation by digestive enzymes and bacteria is limited. Using human cells, tissues and S9 fractions, we were able to demonstrate that small intestine and liver possess strong deacetylation capacity compared to colon and kidneys. Interestingly, in most cases, deacetylation was more efficient for 3ADON than 15ADON. Although we initially thought that carboxylesterases (CES) could be responsible for the deacetylation of 3/15ADON, the use of pure human CES1/2 and of CES inhibitor demonstrated that CES are not involved. Taken together, our original model system allowed us to identify the small intestine and the liver as the main site of deacetylation of ingested 3/15ADON in humans. PMID:27483321

  13. B-esterase activities and blood cell morphology in the frog Leptodactylus chaquensis (Amphibia: Leptodactylidae) on rice agroecosystems from Santa Fe Province (Argentina).

    Science.gov (United States)

    Attademo, Andrés M; Cabagna-Zenklusen, Mariana; Lajmanovich, Rafael C; Peltzer, Paola M; Junges, Celina; Bassó, Agustín

    2011-01-01

    Activity of B-esterases (BChE: butyrylcholinesterase and CbE: carboxylesterase using two model substrates: α-naphthyl acetate and 4-nitrophenyl valerate) in a native frog, Leptodactylus chaquensis from rice fields (RF1: methamidophos and RF2: cypermethrin and endosulfan sprayed by aircraft) and non-contaminated area (pristine forest) was measured. The ability of pyridine-2-aldoxime methochloride (2-PAM) to reactivate BChE levels was also explored. In addition, changes in blood cell morphology and parasite infection were determined. Mean values of plasma BChE activities were lower in samples from the two rice fields than in those from the reference site. CbE (4-nitrophenyl valerate) levels varied in the three sites studied, being highest in RF1. Frog plasma from RF1 showed positive reactivation of BChE activity after incubation with 2-PAM. Blood parameters of frogs from RF2 revealed morphological alterations (anisochromasia and immature erythrocytes frequency). Moreover, a major infection of protozoan Trypanosoma sp. in individuals from the two rice fields was detected. We suggest that integrated use of several biomarkers (BChE and CBEs, chemical reactivation of plasma with 2-PAM, and blood cell parameters) may be a promising procedure for use in biomonitoring programmes to diagnose pesticide exposure of wild populations of this frog and other native anuran species in Argentina.

  14. Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris

    Energy Technology Data Exchange (ETDEWEB)

    Collange, B. [Universite d' Avignon et des Pays de Vaucluse, UMR 406 Abeilles et Environnement, Site AGROPARC, F-84914, Avignon Cede 09 (France); Wheelock, C.E. [Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 171 77, Stockholm (Sweden); Rault, M.; Mazzia, C. [Universite d' Avignon et des Pays de Vaucluse, UMR 406 Abeilles et Environnement, Site AGROPARC, F-84914, Avignon Cede 09 (France); Capowiez, Y. [INRA, Unite PSH, Site AGROPARC, F-84914 Avignon Cedex 09 (France); Sanchez-Hernandez, J.C., E-mail: juancarlos.sanchez@uclm.e [Laboratory of Ecotoxicology, Faculty of Environmental Science, University of Castilla-La Mancha, Avda. Carlos III s/n, 45071, Toledo (Spain)

    2010-06-15

    Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg{sup -1} chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (<=1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field. - Esterase inhibition combined with oxime reactivation methods is a suitable approach for monitoring organophosphate contamination

  15. A Francisella virulence factor catalyses an essential reaction of biotin synthesis.

    Science.gov (United States)

    Feng, Youjun; Napier, Brooke A; Manandhar, Miglena; Henke, Sarah K; Weiss, David S; Cronan, John E

    2014-01-01

    We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side-chain. Expression of bioJ allows growth of an Escherichia coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel subclade of the α/β-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted reaction in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.

  16. Acute toxicity and sublethal effects of fipronil on detoxification enzymes in juvenile zebrafish (Danio rerio).

    Science.gov (United States)

    Wu, Haihua; Gao, Cuie; Guo, Yaping; Zhang, Yuping; Zhang, Jianzhen; Ma, Enbo

    2014-10-01

    The acute toxicity of fipronil and its sublethal effects on detoxification enzymes (carboxylesterases (CarEs), glutathione S-transferases (GSTs), and 7-ethoxycoumarin O-deethylase (ECOD)) in zebrafish (Danio rerio) were investigated. The results indicated that the 24-h LC50 of fipronil for zebrafish was 220.4 μg/L (95% CI: 173.7-272.4 μg/L). Sublethal concentrations of fipronil did not cause significant changes in CarEs activities. In the liver and muscle tissues, GST activities at the tested concentrations did not significantly differ from those in the control. In the brain and gill tissues, GST activities at a concentration of 4 μg/L were significantly lower than those at a concentration of 2 μg/L. The results suggest that CarEs and GSTs were not suitable biomarkers for fipronil effects in D. rerio. A significant induction in the ECOD activities in the brain, gill, liver, and muscle tissues was observed compared with the control. Moreover, the dose-dependent responses of the ECOD activity were observed after treatment with sublethal concentrations of fipronil in the range of 2-20 μg/L. The results suggested that ECOD could be a suitable biomarker of fipronil effects in D. rerio.

  17. Draft genome of the most devastating insect pest of coffee worldwide: the coffee berry borer, Hypothenemus hampei

    KAUST Repository

    Vega, Fernando E.

    2015-07-31

    The coffee berry borer, Hypothenemus hampei, is the most economically important insect pest of coffee worldwide. We present an analysis of the draft genome of the coffee berry borer, the third genome for a Coleopteran species. The genome size is ca. 163 Mb with 19,222 predicted protein-coding genes. Analysis was focused on genes involved in primary digestion as well as gene families involved in detoxification of plant defense molecules and insecticides, such as carboxylesterases, cytochrome P450, gluthathione S-transferases, ATP-binding cassette transporters, and a gene that confers resistance to the insecticide dieldrin. A broad range of enzymes capable of degrading complex polysaccharides were identified. We also evaluated the pathogen defense system and found homologs to antimicrobial genes reported in the Drosophila genome. Ten cases of horizontal gene transfer were identified with evidence for expression, integration into the H. hampei genome, and phylogenetic evidence that the sequences are more closely related to bacterial rather than eukaryotic genes. The draft genome analysis broadly expands our knowledge on the biology of a devastating tropical insect pest and suggests new pest management strategies.

  18. Accurate protein structure annotation through competitive diffusion of enzymatic functions over a network of local evolutionary similarities.

    Science.gov (United States)

    Venner, Eric; Lisewski, Andreas Martin; Erdin, Serkan; Ward, R Matthew; Amin, Shivas R; Lichtarge, Olivier

    2010-12-13

    High-throughput Structural Genomics yields many new protein structures without known molecular function. This study aims to uncover these missing annotations by globally comparing select functional residues across the structural proteome. First, Evolutionary Trace Annotation, or ETA, identifies which proteins have local evolutionary and structural features in common; next, these proteins are linked together into a proteomic network of ETA similarities; then, starting from proteins with known functions, competing functional labels diffuse link-by-link over the entire network. Every node is thus assigned a likelihood z-score for every function, and the most significant one at each node wins and defines its annotation. In high-throughput controls, this competitive diffusion process recovered enzyme activity annotations with 99% and 97% accuracy at half-coverage for the third and fourth Enzyme Commission (EC) levels, respectively. This corresponds to false positive rates 4-fold lower than nearest-neighbor and 5-fold lower than sequence-based annotations. In practice, experimental validation of the predicted carboxylesterase activity in a protein from Staphylococcus aureus illustrated the effectiveness of this approach in the context of an increasingly drug-resistant microbe. This study further links molecular function to a small number of evolutionarily important residues recognizable by Evolutionary Tracing and it points to the specificity and sensitivity of functional annotation by competitive global network diffusion. A web server is at http://mammoth.bcm.tmc.edu/networks.

  19. Pharmacokinetic Modeling and Monte Carlo Simulation to Predict Interindividual Variability in Human Exposure to Oseltamivir and Its Active Metabolite, Ro 64-0802.

    Science.gov (United States)

    Ito, Mototsugu; Kusuhara, Hiroyuki; Ose, Atsushi; Kondo, Tsunenori; Tanabe, Kazunari; Nakayama, Hideki; Horita, Shigeru; Fujita, Takuya; Sugiyama, Yuichi

    2017-01-01

    Oseltamivir (Tamiflu®) is a prodrug of Ro 64-0802, a selective inhibitor of influenza virus neuraminidase. There is a possible relationship between oseltamivir treatment and neuropsychiatric adverse events; although this has not been established, close monitoring is recommended on the prescription label. The objective of this study was to predict interindividual variability of human exposure to oseltamivir and its active metabolite Ro 64-0802. By leveraging mathematical models and computations, physiological parameters in virtual subjects were generated with population means and coefficient of variations collected from the literature or produced experimentally. Postulated functional changes caused by genetic mutations in four key molecules, carboxylesterase 1A1, P-glycoprotein, organic anion transporter 3, and multidrug resistance-associated protein 4, were also taken into account. One hundred thousand virtual subjects were generated per simulation, which was iterated 20 times with different random number generator seeds. Even in the most exaggerated case, the systemic areas under the concentration-time curve (AUCs) of oseltamivir and Ro 64-0802 were increased by at most threefold compared with the population mean. By contrast, the brain AUCs of oseltamivir and Ro 64-0802 were increased up to about sevenfold and 40-fold, respectively, compared with the population means. This unexpectedly high exposure to oseltamivir or Ro 64-0802, which occurs extremely rarely, might trigger adverse central nervous system effects in the clinical setting.

  20. RNA interference: Applications and advances in insect toxicology and insect pest management.

    Science.gov (United States)

    Kim, Young Ho; Soumaila Issa, Moustapha; Cooper, Anastasia M W; Zhu, Kun Yan

    2015-05-01

    Since its discovery, RNA interference (RNAi) has revolutionized functional genomic studies due to its sequence-specific nature of post-transcriptional gene silencing. In this paper, we provide a comprehensive review of the recent literature and summarize the current knowledge and advances in the applications of RNAi technologies in the field of insect toxicology and insect pest management. Many recent studies have focused on identification and validation of the genes encoding insecticide target proteins, such as acetylcholinesterases, ion channels, Bacillus thuringiensis receptors, and other receptors in the nervous system. RNAi technologies have also been widely applied to reveal the role of genes encoding cytochrome P450 monooxygenases, carboxylesterases, and glutathione S-transferases in insecticide detoxification and resistance. More recently, studies have focused on understanding the mechanism of insecticide-mediated up-regulation of detoxification genes in insects. As RNAi has already shown great potentials for insect pest management, many recent studies have also focused on host-induced gene silencing, in which several RNAi-based transgenic plants have been developed and tested as proof of concept for insect pest management. These studies indicate that RNAi is a valuable tool to address various fundamental questions in insect toxicology and may soon become an effective strategy for insect pest management.

  1. Identification of a major Quantitative Trait Locus determining resistance to the organophosphate temephos in the dengue vector mosquito Aedes aegypti.

    Science.gov (United States)

    Paiva, Marcelo H S; Lovin, Diane D; Mori, Akio; Melo-Santos, Maria A V; Severson, David W; Ayres, Constância F J

    2016-01-01

    Organophosphate insecticides (OP) have extensively been used to control mosquitoes, such as the vector Aedes aegypti. Unfortunately, OP resistance has hampered control programs worldwide. We used Quantitative Trait Locus (QTL) mapping to evaluate temephos resistance in two F1 intercross populations derived from crosses between a resistant Ae. aegypti strain (RecR) and two susceptible strains (MoyoD and Red). A single major effect QTL was identified on chromosome 2 of both segregating populations, named rtt1 (resistance to temephos 1). Bioinformatics analyses identified a cluster of carboxylesterase genes (CCE) within the rtt1 interval. qRT-PCR demonstrated that different CCEs were up-regulated in F2 resistant individuals from both crosses. However, none exceeded the 2-fold expression. Primary mechanisms for temephos resistance may vary between Ae. aegypti populations, yet also appear to support previous findings suggesting that multiple linked esterase genes may contribute to temephos resistance in the RecR strain as well as other populations.

  2. Activity of esterases from different tissues of freshwater fish and responses of their isoenzymes to inhibitors.

    Science.gov (United States)

    Li, S N; Fan, D F

    1997-06-06

    Activity of nonspecific esterase from different tissues (i.e., liver, gallbladder, heart, intestine, and muscle) of five species of freshwater fish, namely, topmouth gudgeon (Pseudorasbora parva), goldfish (Carassius auratus), nile tilapia (Tilapia nilotica), mosquitofish (Gambusia affinis), and rainbow trout (Salmo gairdneri) was tested using alpha-naphthyl acetate as substrate. The results indicated that activity of the enzyme was mainly concentrated in the digestive system (i.e., intestine, liver, bile). The overall activity was highest in nile tilapia, followed by mosquitofish, topmouth gudgeon, goldfish, and lowest in rainbow trout. Electrophoresis and the following in vitro treatment of the isoenzymes with triphenol phosphate (TPP, an inhibitor of carboxylesterase) indicated the TPP-sensitive esterase was mainly distributed in liver of the five species. The enzyme was not found in the other five tissues (including gill) except in gallbladder of topmouth gudgeon and goldfish. The correlation was obviously improved between susceptibility and detoxification capacity if activity of the TPP-sensitive esterase was employed instead of that of the nonspecific esterase to make the comparison. In vitro treatment of nonspecific esterase in liver with malaoxon proved that the active metabolite of malathion inhibited a different isoenzyme from the TPP-sensitive one.

  3. A Linkage Map and QTL Analysis for Pyrethroid Resistance in the Bed Bug Cimex lectularius

    Science.gov (United States)

    Fountain, Toby; Ravinet, Mark; Naylor, Richard; Reinhardt, Klaus; Butlin, Roger K.

    2016-01-01

    The rapid evolution of insecticide resistance remains one of the biggest challenges in the control of medically and economically important pests. Insects have evolved a diverse range of mechanisms to reduce the efficacy of the commonly used classes of insecticides, and finding the genetic basis of resistance is a major aid to management. In a previously unstudied population, we performed an F2 resistance mapping cross for the common bed bug, Cimex lectularius, for which insecticide resistance is increasingly widespread. Using 334 SNP markers obtained through RAD-sequencing, we constructed the first linkage map for the species, consisting of 14 putative linkage groups (LG), with a length of 407 cM and an average marker spacing of 1.3 cM. The linkage map was used to reassemble the recently published reference genome, facilitating refinement and validation of the current genome assembly. We detected a major QTL on LG12 associated with insecticide resistance, occurring in close proximity (1.2 Mb) to a carboxylesterase encoding candidate gene for pyrethroid resistance. This provides another example of this candidate gene playing a major role in determining survival in a bed bug population following pesticide resistance evolution. The recent availability of the bed bug genome, complete with a full list of potential candidate genes related to insecticide resistance, in addition to the linkage map generated here, provides an excellent resource for future research on the development and spread of insecticide resistance in this resurging pest species. PMID:27733453

  4. A Linkage Map and QTL Analysis for Pyrethroid Resistance in the Bed Bug Cimex lectularius

    Directory of Open Access Journals (Sweden)

    Toby Fountain

    2016-12-01

    Full Text Available The rapid evolution of insecticide resistance remains one of the biggest challenges in the control of medically and economically important pests. Insects have evolved a diverse range of mechanisms to reduce the efficacy of the commonly used classes of insecticides, and finding the genetic basis of resistance is a major aid to management. In a previously unstudied population, we performed an F2 resistance mapping cross for the common bed bug, Cimex lectularius, for which insecticide resistance is increasingly widespread. Using 334 SNP markers obtained through RAD-sequencing, we constructed the first linkage map for the species, consisting of 14 putative linkage groups (LG, with a length of 407 cM and an average marker spacing of 1.3 cM. The linkage map was used to reassemble the recently published reference genome, facilitating refinement and validation of the current genome assembly. We detected a major QTL on LG12 associated with insecticide resistance, occurring in close proximity (1.2 Mb to a carboxylesterase encoding candidate gene for pyrethroid resistance. This provides another example of this candidate gene playing a major role in determining survival in a bed bug population following pesticide resistance evolution. The recent availability of the bed bug genome, complete with a full list of potential candidate genes related to insecticide resistance, in addition to the linkage map generated here, provides an excellent resource for future research on the development and spread of insecticide resistance in this resurging pest species.

  5. Radiosynthesis of a novel potential adenosine A{sub 3} receptor ligand, 5-ethyl 2,4-diethyl-3-((2-[{sup 18}F]fluoroethyl)sulfanylcarbonyl)-6-phenylpyridine-5-carboxylate ([{sup 18}F]FE rate at SUPPY:2)

    Energy Technology Data Exchange (ETDEWEB)

    Haeusler, D. [Dept. of Nuclear Medicine, Medical Univ. of Vienna (Austria); Dept. of Pharmaceutical Tech. and Biopharmaceutics, Univ. of Vienna (Austria); Mitterhauser, M. [Dept. of Nuclear Medicine, Medical Univ. of Vienna (Austria); Dept. of Pharmaceutical Tech. and Biopharmaceutics, Univ. of Vienna (Austria); Hospital Pharmacy of the General Hospital of Vienna (Austria); Mien, L.K. [Dept. of Nuclear Medicine, Medical Univ. of Vienna (Austria); Dept. of Pharmaceutical Tech. and Biopharmaceutics, Univ. of Vienna (Austria); Dept. of Psychiatry and Psychotherapy, Medical Univ. of Vienna (Austria); Shanab, K.; Spreitzer, H. [Dept. of Drug and Natural Product Synthesis, Univ. of Vienna (Austria); Lanzenberger, R.R [Dept. of Psychiatry and Psychotherapy, Medical Univ. of Vienna (Austria); Schirmer, E. [Dept. of Nuclear Medicine, Medical Univ. of Vienna (Austria); Dept. of Drug and Natural Product Synthesis, Univ. of Vienna (Austria); Ungersboeck, J.; Wadsak, W. [Dept. of Nuclear Medicine, Medical Univ. of Vienna (Austria); Dept. of Inorganic Chemistry, Univ. of Vienna (Austria); Nics, L. [Dept. of Nuclear Medicine, Medical Univ. of Vienna (Austria); Dept. of Nutritional Sciences, Univ. of Vienna (Austria); Viernstein, H. [Dept. of Pharmaceutical Tech. and Biopharmaceutics, Univ. of Vienna (Austria); Dudezak, R.; Kletter, K. [Dept. of Nuclear Medicine, Medical Univ. of Vienna (Austria)

    2009-07-01

    Since, to date very limited information on the distribution and function of the adenosine A{sub 3} receptor is available, the development of suitable radioligands is needed. Recently, we introduced [{sup 18}F]FE rate at SUPPY (5-(2-[{sup 18}F]fluoroethyl) 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate) as the first PET-ligand for the A3R. Regarding the metabolic profile - this class of dialkylpyridines comprises two ester functions within one molecule, one carboxylic and one thiocarboxylic - one could expect carboxylesterases significantly contributing to cleavage and degradation. Therefore, our aim was the development of [{sup 18}F]FE rate at SUPPY:2 (5-ethyl 2,4-diethyl-3-((2-[{sup 18}F]fluoroethyl)sulfanylcarbonyl)-6-phenylpyridine-5-carboxylate), the functional isomer containing the label at the thiocarboxylic moiety. For satisfactory yields in high scale radiosyntheses, a reaction temperature of 75 C has to be applied for at least 20 min using 20 mg/mL of precursor. So far, 6 complete high-scale radiosyntheses were performed. Starting from an average of 51.2 {+-} 21.8 GBq (mean{+-}SD) [{sup 18}F]fluoride, 5.8 {+-} 4.1 GBq of formulated [{sup 18}F]FE rate at SUPPY:2 (12.0{+-}5.4%, based on [{sup 18}F]fluoride, not corrected for decay) were prepared in 75 {+-} 8 min. (orig.)

  6. Role of kdr and esterase-mediated metabolism in pyrethroid-resistant populations of Haematobia irritans irritans (Diptera: Muscidae) in Brazil.

    Science.gov (United States)

    Guerrero, Felix D; Barros, A Thadeu M

    2006-09-01

    The horn fly, Haematobia irritans irritans (L.) (Diptera: Muscidae), has become a problem for Brazilian cattle producers even though its introduction into Brazil is relatively recent. Failure to control this cattle pest is becoming a concern, and horn fly populations from several ranches from the state of Mato Grosso do Sul were surveyed for pyrethroid resistance. Susceptibility bioassays revealed that cypermethrin resistance was widespread and reached high levels in horn fly populations throughout the state, with resistance factors (RFs) ranging from 50.4 to 704.8. Synergist bioassays failed to detect a major role for esterases as a pyrethroid resistance mechanism in these populations, except for the highly pyrethroid-resistant Estrela do Oeste population (RF = 704.8). The kdr sodium channel gene mutation was not detected in eight of the 13 populations, but Oeste exhibited this mutation. Neither the superkdr sodium channel gene mutation nor a resistance-associated gene mutation in the HialphaE7 carboxylesterase were found in any of the fly populations. Although target site insensitivity (kdr) and esterase-mediated metabolism occur in horn fly populations from Mato Grosso do Sul state, it seems that they are not the major mechanism causing pyrethroid resistance in most of these populations.

  7. Biochemical response to exposure to six textile dyes in early developmental stages of Xenopus laevis.

    Science.gov (United States)

    Güngördü, Abbas; Birhanli, Ayse; Ozmen, Murat

    2013-01-01

    The present study was undertaken to determine the toxic effect of a lethal concentration of six different commercially used textile dyes on the 46th stage of Xenopus laevis tadpoles. The tadpoles were exposed to Astrazon Red FBL, Astrazon Blue FGRL, Remazol Red RR, Remazol Turquoise Blue G-A, Cibacron Red FN-3G, and Cibacron Blue FN-R for 168 h in static test conditions, and thus, 168-h median lethal concentrations (LC(50)s) of each dye were determined to be 0.35, 0.13, 112, 7, 359, and 15.8 mg/L, respectively. Also, to evaluate the sublethal effects of each dye, tadpoles were exposed to different concentrations of dyes (with respect to 168-h LC(50)s) for 24 h. The alteration of selected enzyme activities was tested. For this aim, glutathione S-transferase (GST), carboxylesterase, and lactate dehydrogenase (LDH) were assayed. After dye exposure, the GST induction or inhibition and LDH induction indicated some possible mechanisms of oxidative stress and deterioration in aerobic respiration processes induced by the tested dyes. Findings of the study suggest that selected biomarker enzymes are useful in understanding the toxic mechanisms of these dyes in X. laevis tadpoles as early warning indicators. Therefore, these selected biomarkers may evaluate the effect of environmental factors, such as textile dye effluents and other industrial pollutants, on amphibians in biomonitoring studies.

  8. Proteins with an alpha/beta hydrolase fold: Relationships between subfamilies in an ever-growing superfamily.

    Science.gov (United States)

    Lenfant, Nicolas; Hotelier, Thierry; Bourne, Yves; Marchot, Pascale; Chatonnet, Arnaud

    2013-03-25

    Alpha/beta hydrolases function as hydrolases, lyases, transferases, hormone precursors or transporters, chaperones or routers of other proteins. The amount of structural and functional available data related to this protein superfamily expands exponentially, as does the number of proteins classified as alpha/beta hydrolases despite poor sequence similarity and lack of experimental data. However the superfamily can be rationally divided according to sequence or structural homologies, leading to subfamilies of proteins with potentially similar functions. Since the discovery of proteins homologous to cholinesterases but devoid of enzymatic activity (e.g., the neuroligins), divergent functions have been ascribed to members of other subfamilies (e.g., lipases, dipeptidylaminopeptidase IV, etc.). To study the potentially moonlighting properties of alpha/beta hydrolases, the ESTHER database (for ESTerase and alpha/beta Hydrolase Enzymes and Relatives; http://bioweb.ensam.inra.fr/esther), which collects, organizes and disseminates structural and functional information related to alpha/beta hydrolases, has been updated with new tools and the web server interface has been upgraded. A new Overall Table along with a new Tree based on HMM models has been included to tentatively group subfamilies. These tools provide starting points for phylogenetic studies aimed at pinpointing the origin of duplications leading to paralogous genes (e.g., acetylcholinesterase versus butyrylcholinesterase, or neuroligin versus carboxylesterase). Another of our goals is to implement new tools to distinguish catalytically active enzymes from non-catalytic proteins in poorly studied or annotated subfamilies.

  9. Hepatic and branchial xenobiotic biomarker responses in Solea spp. from several NW Mediterranean fishing grounds.

    Science.gov (United States)

    Siscar, R; Varó, I; Solé, M

    2015-12-01

    The common sole, Solea solea and the Senegalese sole, Solea senegalensis are two important commercial benthic species that coexist in the NW Mediterranean Sea. Several common biomarkers of chemical exposure were measured in two organs (liver and gills) involved in a different degree in biotransformation and detoxification processes. These parameters were: phase I cytochrome P450 CYP1A-dependent ethoxyresorufin O-deethylase and carboxylesterase activities, phase II glutathione S-transferase activity and the enzymatic antioxidants: catalase, glutathione reductase and glutathione peroxidase. Principal Component Analysis (PCA) considering biometric variables (size and weight) and all liver and gill biomarkers discriminated at a certain extent individuals of both species collected at the different fishing grounds. Esterase inhibition by the organophosphorus pesticides dichlorvos and diazinon was also compared in vitro in muscle, liver and gill of the two species revealing a differential sensitivity. The use of benthic sole in pollution monitoring of Southern Europe is discussed as local sentinel in respect to other benthic fish from more Northern latitudes.

  10. Critical role of neutral cholesteryl ester hydrolase 1 in cholesteryl ester hydrolysis in murine macrophages[S

    Science.gov (United States)

    Sakai, Kent; Igarashi, Masaki; Yamamuro, Daisuke; Ohshiro, Taichi; Nagashima, Shuichi; Takahashi, Manabu; Enkhtuvshin, Bolormaa; Sekiya, Motohiro; Okazaki, Hiroaki; Osuga, Jun-ichi; Ishibashi, Shun

    2014-01-01

    Hydrolysis of intracellular cholesteryl ester (CE) is the rate-limiting step in the efflux of cholesterol from macrophage foam cells. In mouse peritoneal macrophages (MPMs), this process is thought to involve several enzymes: hormone-sensitive lipase (Lipe), carboxylesterase 3 (Ces3), neutral CE hydrolase 1 (Nceh1). However, there is some disagreement over the relative contributions of these enzymes. To solve this problem, we first compared the abilities of several compounds to inhibit the hydrolysis of CE in cells overexpressing Lipe, Ces3, or Nceh1. Cells overexpressing Ces3 had negligible neutral CE hydrolase activity. We next examined the effects of these inhibitors on the hydrolysis of CE and subsequent cholesterol trafficking in MPMs. CE accumulation was increased by a selective inhibitor of Nceh1, paraoxon, and two nonselective inhibitors of Nceh1, (+)-AS115 and (−)-AS115, but not by two Lipe-selective inhibitors, orlistat and 76-0079. Paraoxon inhibited cholesterol efflux to apoA-I or HDL, while 76-0079 did not. These results suggest that Nceh1 plays a dominant role over Lipe in the hydrolysis of CE and subsequent cholesterol efflux in MPMs. PMID:24868095

  11. Effects of dietary nickel on detoxification enzyme activities in the midgut of Spodoptera litura Fabricius larvae

    Institute of Scientific and Technical Information of China (English)

    SUN HongXia; ZHOU Qiang; TANG WenCheng; SHU YingHua; ZHANG GuRen

    2008-01-01

    Nickel accumulated in midugt of Spodoptera litura Fabricius could induce the expression of metal-Iothionein, one of the most important detoxification proteins in organisms. In the present study, the effects of dietary nickel on the activities of detoxification enzymes, such as carboxylesterase (CarE) and glutathione S-transferase (GST) in the midgut of S. litura larvae have been studied to get an un-derstanding of the detoxification mechanisms of S. litura larvae to excessive nickel. Results showed that CarE activities in the midgut of the 5th instar larvae decreased at lower levels of nickel (≤5 mg/kg), while increased with increasing nickel doses at higher levels of nickel (≥10 mg/kg) exposure in suc-cessive 3 generations. CarE activities of the 6th instar larvae were also characterized as inhibited at low levels of nickel exposure, and improved at higher levels in the 1st generation. CarE activities of 6th instar larvae in the 2rid and 3rd generations were all lower than that in control. However, GST activities in the midgut of the 5th and 6th instar larvae all increased with increasing nickel doses (1-20 mg/kg) in diets.

  12. Insecticide resistance in Bemisia tabaci from Cyprus

    Institute of Scientific and Technical Information of China (English)

    Vassilis Vassiliou; Maria Emmanouilidou; Andreas Perrakis; Evangelia Morou; John Vontas; Anastasia Tsagkarakou; Emmanouil Roditakis

    2011-01-01

    A comprehensive study on the Bemisia tabaci(biotype B)resistance to neonicotinoid insecticides imidacloprid,acetamiprid and thiamethoxam,and pyrethroid bifenthrin was conducted in Cyprus.The resistance level to eight field-collected B.tabaci populations was investigated.The activities of enzymes involved in metabolic detoxification and the frequencies of pyrethroid and organophosphates target site resistance mutations were determined.Moderate to high levels of resistance were detected for imidacloprid(resistance factor[RF]77-392)and thiamethoxam(RF 50-164)while low resistance levels were observed for acetamiprid(RF 7-12).Uniform responses by the Cypriot whiteflies could be observed against all neonicotinoid insecticides.No cross-resistance between the neonicotinoids was detected as well as no association with the activity of the P450 microsomal oxidases.Only imidacloprid resistance correlated with carboxylesterase activity.Low to extremely high resistance was observed for insecticide bifenthrin(RF 49-1 243)which was associated with the frequency of the resistant allele in the sodium channel gene but not with the activity of the detoxification enzymes.Finally,the F331W mutation in the acetylcholinesterase enzyme ace1 gene was fixed in all B.tabaci populations from Cyprus.

  13. Copy Number Variation of UGT 2B Genes in Indian Families Using Whole Genome Scans

    Directory of Open Access Journals (Sweden)

    Avinash M. Veerappa

    2016-01-01

    Full Text Available Background and Objectives. Uridine diphospho-glucuronosyltransferase 2B (UGT2B is a family of genes involved in metabolizing steroid hormones and several other xenobiotics. These UGT2B genes are highly polymorphic in nature and have distinct polymorphisms associated with specific regions around the globe. Copy number variations (CNVs status of UGT2B17 in Indian population is not known and their disease associations have been inconclusive. It was therefore of interest to investigate the CNV profile of UGT2B genes. Methods. We investigated the presence of CNVs in UGT2B genes in 31 members from eight Indian families using Affymetrix Genome-Wide Human SNP Array 6.0 chip. Results. Our data revealed >50% of the study members carried CNVs in UGT2B genes, of which 76% showed deletion polymorphism. CNVs were observed more in UGT2B17 (76.4% than in UGT2B15 (17.6%. Molecular network and pathway analysis found enrichment related to steroid metabolic process, carboxylesterase activity, and sequence specific DNA binding. Interpretation and Conclusion. We report the presence of UGT2B gene deletion and duplication polymorphisms in Indian families. Network analysis indicates the substitutive role of other possible genes in the UGT activity. The CNVs of UGT2B genes are very common in individuals indicating that the effect is neutral in causing any suspected diseases.

  14. Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines.

    Science.gov (United States)

    Ye, Fei; Xin, Zhongshuai; Han, Wei; Fan, Jingjing; Yin, Bin; Wu, Shuzhen; Yang, Wei; Yuan, Jiangang; Qiang, Boqin; Sun, Wei; Peng, Xiaozhong

    2015-01-01

    Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

  15. RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding.

    Science.gov (United States)

    Turner, C T; Davy, M W; MacDiarmid, R M; Plummer, K M; Birch, N P; Newcomb, R D

    2006-06-01

    RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene (EposCXE1) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene (EposPBP1) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of EposPBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed.

  16. B-esterase determination and organophosphate insecticide inhibitory effects in JEG-3 trophoblasts.

    Science.gov (United States)

    Espinoza, Marlon; Rivero Osimani, Valeria; Sánchez, Victoria; Rosenbaum, Enrique; Guiñazú, Natalia

    2016-04-01

    The placenta and trophoblasts express several B-esterases. This family includes acethylcholinesterase (AChE), carboxylesterase (CES) and butyrylcholinesterase (BChE), which are important targets of organophosphate insecticide (OP) toxicity. To better understand OP effects on trophoblasts, B-esterase basal activity and kinetic behavior were studied in JEG-3 choriocarcinoma cell cultures. Effects of the OP azinphos-methyl (Am) and chlorpyrifos (Cp) on cellular enzyme activity were also evaluated. JEG-3 cells showed measurable activity levels of AChE and CES, while BChE was undetected. Recorded Km for AChE and CES were 0.33 and 0.26 mM respectively. Native gel electrophoresis and RT-PCR analysis demonstrated CES1 and CES2 isoform expression. Cells exposed for 4 and 24 h to the OP Am or Cp, showed a differential CES and AChE inhibition profiles. Am inhibited CES and AChE at 4 h treatment while Cp showed the highest inhibition profile at 24 h. Interestingly, both insecticides differentially affected CES1 and CES2 activities. Results demonstrated that JEG-3 trophoblasts express AChE, CES1 and CES2. B-esterase enzymes were inhibited by in vitro OP exposure, indicating that JEG-3 cells metabolization capabilities include phase I enzymes, able to bioactivate OP. In addition, since CES enzymes are important for medicinal drug activation/deactivation, OP exposure may interfere with trophoblast CES metabolization, probably being relevant in a co-exposure scenario during pregnancy.

  17. Determination of biomarkers for polycyclic aromatic hydrocarbons (PAHs) toxicity to earthworm (Eisenia fetida).

    Science.gov (United States)

    Nam, Tae-Hoon; Jeon, Hwang-Ju; Mo, Hyung-ho; Cho, Kijong; Ok, Yong-Sik; Lee, Sung-Eun

    2015-12-01

    Polycyclic aromatic hydrocarbon (PAH) compounds are persistent, carcinogenic, and mutagenic. When PAHs enter agricultural soils through sewage sludge, they pose an environmental risk to soil organisms, including earthworms. Therefore, we aimed to determine the toxic effects of PAHs on earthworms. Five PAHs were used: fluorene, anthracene, phenanthrene, fluoranthene, and pyrene. Only fluorene and phenanthrene exhibited toxicity (LC50 values 394.09 and 114.02 g L(-1), respectively) against the earthworm Eisenia fetida. None of the other PAHs tested in this study enhanced the mortality of adult earthworm until the concentrations reached to 1000 g L(-1). After exposure to PAHs, acetylcholinesterase (AChE) activity in E. fetida decreased in a concentration-dependent manner, and phenanthrene exhibited the strongest inhibitory effect on AChE, followed by fluorene. Activity of a representative detoxifying enzyme, carboxylesterase, was dramatically reduced in E. fetida exposed to all tested PAHs in comparison with that observed in the control test. The remaining glutathione S-transferase activity significantly decreased in E. fetida after exposure to PAHs. To profile small proteins PAHs tested in E. fetida.

  18. Use of 3-[18F]fluoropropanesulfonyl chloride as a prosthetic agent for the radiolabelling of amines: Investigation of precursor molecules, labelling conditions and enzymatic stability of the corresponding sulfonamides

    Directory of Open Access Journals (Sweden)

    Reik Löser

    2013-05-01

    Full Text Available 3-[18F]Fluoropropanesulfonyl chloride, a recently proposed prosthetic agent for fluorine-18 labelling, was prepared in a two-step radiosynthesis via 3-[18F]fluoropropyl thiocyanate as an intermediate. Two benzenesulfonate-based radiolabelling precursors were prepared by various routes. Comparing the reactivities of 3-thiocyanatopropyl nosylate and the corresponding tosylate towards [18F]fluoride the former proved to be superior accounting for labelling yields of up to 85%. Conditions for a reliable transformation of 3-[18F]fluoropropyl thiocyanate to the corresponding sulfonyl chloride with the potential for automation have been identified. The reaction of 3-[18F]fluoropropanesulfonyl chloride with eight different aliphatic and aromatic amines was investigated and the identity of the resulting 18F-labelled sulfonamides was confirmed chromatographically by comparison with their nonradioactive counterparts. Even for weakly nucleophilic amines such as 4-nitroaniline the desired radiolabelled sulfonamides were accessible in satisfactory yields owing to systematic variation of the reaction conditions. With respect to the application of the 18F-fluoropropansulfonyl group to the labelling of compounds relevant as imaging agents for positron emission tomography (PET, the stability of N-(4-fluorophenyl-3-fluoropropanesulfonamide against degradation catalysed by carboxylesterase was investigated and compared to that of the analogous fluoroacetamide.

  19. Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil.

    Science.gov (United States)

    Zuo, Zhenqiang; Gong, Ting; Che, You; Liu, Ruihua; Xu, Ping; Jiang, Hong; Qiao, Chuanling; Song, Cunjiang; Yang, Chao

    2015-06-01

    Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P. putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (10(6) cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.

  20. Animal models that best reproduce the clinical manifestations of human intoxication with organophosphorus compounds.

    Science.gov (United States)

    Pereira, Edna F R; Aracava, Yasco; DeTolla, Louis J; Beecham, E Jeffrey; Basinger, G William; Wakayama, Edgar J; Albuquerque, Edson X

    2014-08-01

    The translational capacity of data generated in preclinical toxicological studies is contingent upon several factors, including the appropriateness of the animal model. The primary objectives of this article are: 1) to analyze the natural history of acute and delayed signs and symptoms that develop following an acute exposure of humans to organophosphorus (OP) compounds, with an emphasis on nerve agents; 2) to identify animal models of the clinical manifestations of human exposure to OPs; and 3) to review the mechanisms that contribute to the immediate and delayed OP neurotoxicity. As discussed in this study, clinical manifestations of an acute exposure of humans to OP compounds can be faithfully reproduced in rodents and nonhuman primates. These manifestations include an acute cholinergic crisis in addition to signs of neurotoxicity that develop long after the OP exposure, particularly chronic neurologic deficits consisting of anxiety-related behavior and cognitive deficits, structural brain damage, and increased slow electroencephalographic frequencies. Because guinea pigs and nonhuman primates, like humans, have low levels of circulating carboxylesterases-the enzymes that metabolize and inactivate OP compounds-they stand out as appropriate animal models for studies of OP intoxication. These are critical points for the development of safe and effective therapeutic interventions against OP poisoning because approval of such therapies by the Food and Drug Administration is likely to rely on the Animal Efficacy Rule, which allows exclusive use of animal data as evidence of the effectiveness of a drug against pathologic conditions that cannot be ethically or feasibly tested in humans.

  1. Calibration and validation of a physiologically based model for soman intoxication in the rat, marmoset, guinea pig and pig.

    Science.gov (United States)

    Chen, Kaizhen; Seng, Kok-Yong

    2012-09-01

    A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model has been developed for low, medium and high levels of soman intoxication in the rat, marmoset, guinea pig and pig. The primary objective of this model was to describe the pharmacokinetics of soman after intravenous, intramuscular and subcutaneous administration in the rat, marmoset, guinea pig, and pig as well as its subsequent pharmacodynamic effects on blood acetylcholinesterase (AChE) levels, relating dosimetry to physiological response. The reactions modelled in each physiologically realistic compartment are: (1) partitioning of C(±)P(±) soman from the blood into the tissue; (2) inhibition of AChE and carboxylesterase (CaE) by soman; (3) elimination of soman by enzymatic hydrolysis; (4) de novo synthesis and degradation of AChE and CaE; and (5) aging of AChE-soman and CaE-soman complexes. The model was first calibrated for the rat, then extrapolated for validation in the marmoset, guinea pig and pig. Adequate fits to experimental data on the time course of soman pharmacokinetics and AChE inhibition were achieved in the mammalian models. In conclusion, the present model adequately predicts the dose-response relationship resulting from soman intoxication and can potentially be applied to predict soman pharmacokinetics and pharmacodynamics in other species, including human.

  2. An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440.

    Science.gov (United States)

    Wang, Yuanyuan; Zhang, Chi; Gong, Ting; Zuo, Zhenqiang; Zhao, Fengjie; Fan, Xu; Yang, Chao; Song, Cunjiang

    2015-06-01

    A markerless gene replacement method was adapted by combining a suicide plasmid, pEX18Tc, with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the medium-chain length polyhydroxyalkanoates (PHA(MCL))-producing strain Pseudomonas mendocina NK-01. An NK-01 5-fluorouracil (5-FU) resistant background strain was first constructed by deleting the chromosomal upp gene. The suicide plasmid pEX18Tc, carrying a functional allele of the upp gene of P. mendocina NK-01, was used to construct the vectors to delete the algA (encoding mannose-1-phosphate guanylyltransferase) and phaZ (encoding PHA(MCL) depolymerase) genes, and a 30 kb chromosomal fragment in the 5-FU resistant background host. The genes were removed efficiently from the genome of P. mendocina NK-01 and left a markerless chromosomal mutant. In addition, two exogenous genes were inserted into the phaC1 (PHA(MCL) polymerase) loci of Pseudomonas putida KT-∆UPP simultaneously. Thus, we constructed a genetically stable and marker-free P. putida KT2440 mutant with integrated mpd (encoding methyl parathion hydrolase (MPH)) and pytH (encoding a pyrethroid-hydrolyzing carboxylesterase (PytH)) gene on the chromosome. The upp-based counterselection system could be further adapted for P. mendocina NK-01 and P. putida KT2440 and used for genome reduction and metabolic pathway engineering.

  3. Transesterification of a series of 12 parabens by liver and small-intestinal microsomes of rats and humans.

    Science.gov (United States)

    Fujino, Chieri; Watanabe, Yoko; Uramaru, Naoto; Kitamura, Shigeyuki

    2014-02-01

    Hydrolytic transformation of parabens (4-hydroxybenzoic acid esters; used as antibacterial agents) to 4-hydroxybenzoic acid and alcohols by tissue microsomes is well-known both in vitro and in vivo. Here, we investigated transesterification reactions of parabens catalyzed by rat and human microsomes, using a series of 12 parabens with C1-C12 alcohol side chains. Transesterification of parabens by rat liver and small-intestinal microsomes occurred in the presence of alcohols in the microsomal incubation mixture. Among the 12 parabens, propylparaben was most effectively transesterified by rat liver microsomes with methanol or ethanol, followed by butylparaben. Relatively low activity was observed with longer-side-chain parabens. In contrast, small-intestinal microsomes exhibited higher activity towards moderately long side-chain parabens, and showed the highest activity toward octylparaben. When parabens were incubated with liver or small-intestinal microsomes in the presence of C1-C12 alcohols, ethanol and decanol were most effectively transferred to parabens by rat liver microsomes and small-intestinal microsomes, respectively. Human liver and small-intestinal microsomes also exhibited significant transesterification activities with different substrate specificities, like rat microsomes. Carboxylesterase isoforms, CES1b and CES1c, and CES2, exhibited significant transesterification activity toward parabens, and showed similar substrate specificity to human liver and small-intestinal microsomes, respectively.

  4. Endophytic fungi producing of esterases: evaluation in vitro of the enzymatic activity using pH indicator.

    Science.gov (United States)

    Lisboa, Helen Cristina Fávero; Biasetto, Carolina Rabal; de Medeiros, João Batista; Âraújo, Angela Regina; Silva, Dulce Helena Siqueira; Teles, Helder Lopes; Trevisan, Henrique Celso

    2013-01-01

    A sensitive and efficient colorimetric method was optimized for detection of esterase enzymes produced by endophytic fungi for development of High-Throughput Screening (HTS). The fungi were isolated and obtained previously from plant species of Cerrado and Atlantic Forest located in areas of environmental preservation in the State of Sao Paulo / Brazil, as part of the project "Chemical and biological prospecting endophytic fungi associated to plant species of Cerrado and Atlantic Forest". The compounds ethyl butyrate, ethyl acetate and methyl propionate were used as standards esters which were hydrolyzed by extracellular enzyme from endophytic fungi (EC. 3.1.1.1--carboxyl-esterases) for production of carboxylic acids. Thus, the reduction of the pH increases the protonated indicator concentration (bromothymol blue), changing the color of the reaction medium (from blue to yellow), that can be observed and measured by spectrophotometry at 616 nm. The methodology with acid-base indicator was performed on 13 microorganisms, aiming Periconia atropurpurea as a potential source of esterase for biotransformation of short chain esters. The results also evidenced that this methodology showed to be efficient, fast, cheap, having low consumption of reagents and easy development, and can be applied to screen carboxylic-ester hydrolases in a large number of microorganisms.

  5. Use of enzyme inhibitors to evaluate the conversion pathways of ester and amide prodrugs: a case study example with the prodrug ceftobiprole medocaril.

    Science.gov (United States)

    Eichenbaum, Gary; Skibbe, Jennifer; Parkinson, Andrew; Johnson, Mark D; Baumgardner, Dawn; Ogilvie, Brian; Usuki, Etsuko; Tonelli, Fred; Holsapple, Jeff; Schmitt-Hoffmann, Anne

    2012-03-01

    An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion.

  6. Carbamate and Pyrethroid Resistance in the Akron Strain of Anopheles gambiae

    Science.gov (United States)

    Mutunga, James M.; Anderson, Troy D.; Craft, Derek T.; Gross, Aaron D.; Swale, Daniel R.; Tong, Fan; Wong, Dawn M.; Carlier, Paul R.; Bloomquist, Jeffrey R.

    2015-01-01

    Insecticide resistance in the malaria vector, Anopheles gambiae is a serious problem, epitomized by the multi-resistant Akron strain, originally isolated in the country of Benin. Here we report resistance in this strain to pyrethroids and DDT (13-fold to 35-fold compared to the susceptible G3 strain), but surprisingly little resistance to etofenprox, a compound sometimes described as a “pseudo-pyrethroid.” There was also strong resistance to topically-applied commercial carbamates (45-fold to 81-fold), except for the oximes aldicarb and methomyl. Biochemical assays showed enhanced cytochrome P450 monooxygenase and carboxylesterase activity, but not that of glutathione-S-transferase. A series of substituted α,α,α,-trifluoroacetophenone oxime methylcarbamates were evaluated for enzyme inhibition potency and toxicity against G3 and Akron mosquitoes. The compound bearing an unsubstituted phenyl ring showed the greatest toxicity to mosquitoes of both strains. Low cross resistance in Akron was retained by all analogs in the series. Kinetic analysis of acetylcholinesterase activity and its inhibition by insecticides in the G3 strain showed inactivation rate constants greater than that of propoxur, and against Akron enzyme inactivation rate constants similar to that of aldicarb. However, inactivation rate constants against recombinant human AChE were essentially identical to that of the G3 strain. Thus, the acetophenone oxime carbamates described here, though potent insecticides that control resistant Akron mosquitoes, require further structural modification to attain acceptable selectivity and human safety. PMID:26047119

  7. The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling.

    Science.gov (United States)

    Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

    2013-10-01

    The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

  8. Characterisation of esterases as potential biomarkers of pesticide exposure in the lugworm Arenicola marina (Annelida: Polychaeta)

    Energy Technology Data Exchange (ETDEWEB)

    Hannam, Marie L. [Ecotoxicology and Stress Biology Research Centre, School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth, Devon, PL4 8AA (United Kingdom)], E-mail: marie.hannam@plymouth.ac.uk; Hagger, Josephine A.; Jones, Malcolm B.; Galloway, Tamara S. [Ecotoxicology and Stress Biology Research Centre, School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth, Devon, PL4 8AA (United Kingdom)

    2008-03-15

    Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC{sub 50} of 5 {mu}g l{sup -1} for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 {mu}g l{sup -1} azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F = 2.75, P = 0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F = 15.33, P < 0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species. - This paper provides a preliminary characterisation of esterase enzyme activities in the tissues and body fluids of the sediment dwelling worm Arenicola marina and explores their potential use as biomarkers of organophosphorus pesticide exposure in the marine environment.

  9. Tumour-selective targeting of drug metabolizing enzymes to treat metastatic cancer.

    Science.gov (United States)

    Wierdl, Monika; Tsurkan, Lyudmila; Hatfield, M Jason; Potter, Philip M

    2016-10-01

    Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of ester-containing xenobiotics. This hydrolysis reaction results in the formation of the corresponding carboxylic acid and alcohol. Due to their highly plastic active site, CEs can hydrolyze structurally very distinct and complex molecules. Because ester groups significantly increase the water solubility of compounds, they are frequently used in the pharmaceutical industry to make relatively insoluble compounds more bioavailable. By default, this results in CEs playing a major role in the distribution and metabolism of these esterified drugs. However, this can be exploited to selectively improve compound hydrolysis, and using specific in vivo targeting techniques can be employed to generate enhanced drug activity. Here, we seek to detail the human CEs involved in esterified molecule hydrolysis, compare and contrast these with CEs present in small mammals and describe novel methods to improve drug therapy by specific delivery of CEs to cells in vivo. Finally, we will discuss the development of such approaches for their potential application towards malignant disease.

  10. Insect-resistant mechanism of transgenic triploid of Chinese white poplar

    Institute of Scientific and Technical Information of China (English)

    Yuan Shengliang; Gao Baojia; Zhang Na

    2006-01-01

    The activities of antidotal enzymes and digestive enzymes of Clostera anachoreta (Fabricius) instar larvae,feeding on leaves of three kinds of insect-resistant clones of transgenic triploid of Chinese white poplar,after 4,12,24,48,72 and 96 h,were investigated.The results showed that,feeding on clone 7,the activity of esterase,carboxylesterase,and mixed-function oxidases in the midgut of the larvae was very much decreased.Feeding on clone 10,those results were less than those of clone 7 and there were few changes on the larvae,which fed on clone 26.The changes of the amylase in the midgut of larvae were the same as those described above.However,the activities of glutathione S-transferase and proteinase were complex,increased markedly after 24 h feeding on clone 7,and then declined rapidly.The same changes were taking place on the larvae feeding on clone 10.There were many slight changes in glutathione S-transferase of the larvae,feeding on clone 26;no changes occurred in the proteinases of the midgut.Thus,the antidotal enzymes and digestive enzymes in the midgut of the larvae were inhibited.This may be the main mechanism of the transgenic triploid of Chinese white poplar.

  11. Response Mechanisms of Pine Caterpillar Enzymatic System to Pine Induced Resistance%油松毛虫体内酶系对油松诱导抗性的响应机制

    Institute of Scientific and Technical Information of China (English)

    张雄帅; 周国娜; 高宝嘉

    2014-01-01

    To explore counter-defense response mechanism of pine caterpillar ( Dendrolinus punctatus tabulaeformis Tsai et Liu) to the induced insect-resistance of Chinese pine (Pinus tabulaeformis),the Chinese pine was fed by different amount pine caterpillars to obtain induced insect-resistance in Pingquan of Hebei Province,and main detoxifying enzymes, protective enzymes and digestive enzymes of pine caterpillars fed on the Chinese pine leaf were determined. The results showed that most of enzymes in the pine caterpillars changed obviously after feeding various induced resistant pine leaves. Carboxylesterase,glutathione-transferase,superoxide dismutase,peroxidase and protease activity of the lavae increased significantly with the increasing feeding stimulation. Acetylcholinesterase,lipase and amylase activity of the lavae had some change but no obviously linear variation,and acetylcholinesterase activity had no change. The results indicated that carboxyl esterase,glutathione-s-transferees,superoxide dismutase,peroxidase and protease were important enzymes with that pine caterpillar responded to the pine defense. Within a certain range,the adaptive enzymes activity in the pine caterpillar increased as the increasing of the action strength and time of the pine induced defensive products to pine caterpillar.

  12. Ethylphenidate as a selective dopaminergic agonist and methylphenidate-ethanol transesterification biomarker.

    Science.gov (United States)

    Patrick, Kennerly S; Corbin, Timothy R; Murphy, Cristina E

    2014-12-01

    We review the pharmaceutical science of ethylphenidate (EPH) in the contexts of drug discovery, drug interactions, biomarker for dl-methylphenidate (MPH)-ethanol exposure, potentiation of dl-MPH abuse liability, contemporary "designer drug," pertinence to the newer transdermal and chiral switch MPH formulations, as well as problematic internal standard. d-EPH selectively targets the dopamine transporter, whereas d-MPH exhibits equipotent actions at dopamine and norepinephrine transporters. This selectivity carries implications for the advancement of tailored attention-deficit/hyperactivity disorder (ADHD) pharmacotherapy in the era of genome-based diagnostics. Abuse of dl-MPH often involves ethanol coabuse. Carboxylesterase 1 enantioselectively transesterifies l-MPH with ethanol to yield l-EPH accompanied by significantly increased early exposure to d-MPH and rapid potentiation of euphoria. The pharmacokinetic component of this drug interaction can largely be avoided using dexmethylphenidate (dexMPH). This notwithstanding, maximal potentiated euphoria occurs following dexMPH-ethanol. C57BL/6 mice model dl-MPH-ethanol interactions: an otherwise depressive dose of ethanol synergistically increases dl-MPH stimulation; a substimulatory dose of dl-MPH potentiates a low, stimulatory dose of ethanol; ethanol elevates blood, brain, and urinary d-MPH concentrations while forming l-EPH. Integration of EPH preclinical neuropharmacology with clinical studies of MPH-ethanol interactions provides a translational approach toward advancement of ADHD personalized medicine and management of comorbid alcohol use disorder.

  13. Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

    Science.gov (United States)

    Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

    2016-01-01

    Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

  14. Accurate protein structure annotation through competitive diffusion of enzymatic functions over a network of local evolutionary similarities.

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    Eric Venner

    Full Text Available High-throughput Structural Genomics yields many new protein structures without known molecular function. This study aims to uncover these missing annotations by globally comparing select functional residues across the structural proteome. First, Evolutionary Trace Annotation, or ETA, identifies which proteins have local evolutionary and structural features in common; next, these proteins are linked together into a proteomic network of ETA similarities; then, starting from proteins with known functions, competing functional labels diffuse link-by-link over the entire network. Every node is thus assigned a likelihood z-score for every function, and the most significant one at each node wins and defines its annotation. In high-throughput controls, this competitive diffusion process recovered enzyme activity annotations with 99% and 97% accuracy at half-coverage for the third and fourth Enzyme Commission (EC levels, respectively. This corresponds to false positive rates 4-fold lower than nearest-neighbor and 5-fold lower than sequence-based annotations. In practice, experimental validation of the predicted carboxylesterase activity in a protein from Staphylococcus aureus illustrated the effectiveness of this approach in the context of an increasingly drug-resistant microbe. This study further links molecular function to a small number of evolutionarily important residues recognizable by Evolutionary Tracing and it points to the specificity and sensitivity of functional annotation by competitive global network diffusion. A web server is at http://mammoth.bcm.tmc.edu/networks.

  15. Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines.

    Directory of Open Access Journals (Sweden)

    Fei Ye

    Full Text Available Chronic hepatitis C virus (HCV infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1, Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1, carboxylesterase 1 (CES1, vimentin, Proteasome activator complex subunit1 (PSME1, and Cathepsin B (CTSB were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

  16. Endophytic fungi producing of esterases: evaluation in vitro of the enzymatic activity using pH indicator

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    Helen Cristina Fávero Lisboa

    2013-09-01

    Full Text Available A sensitive and efficient colorimetric method was optimized for detection of esterase enzymes produced by endophytic fungi for development of High-Throughput Screening (HTS. The fungi were isolated and obtained previously from plant species of Cerrado and Atlantic Forest located in areas of environmental preservation in the State of Sao Paulo / Brazil, as part of the project "Chemical and biological prospecting endophytic fungi associated to plant species of Cerrado and Atlantic Forest". The compounds ethyl butyrate, ethyl acetate and methyl propionate were used as standards esters which were hydrolyzed by extracellular enzyme from endophytic fungi (EC. 3.1.1.1 -carboxylesterases for production of carboxylic acids. Thus, the reduction of the pH increases the protonated indicator concentration (bromothymol blue, changing the color of the reaction medium (from blue to yellow, that can be observed and measured by spectrophotometry at 616 nm. The methodology with acid-base indicator was performed on 13 microorganisms, aiming Periconia atropurpurea asapotential source of esterase for biotransformation of short chain esters. The results also evidenced that this methodology showed to be efficient, fast, cheap, having low consumption of reagents and easy development, and can be applied to screen carboxylic-ester hydrolases in a large number of microorganisms.

  17. Identification of biotransformation enzymes in the antennae of codling moth Cydia pomonella.

    Science.gov (United States)

    Huang, Xinglong; Liu, Lu; Su, Xiaoji; Feng, Jinian

    2016-04-10

    Biotransformation enzymes are found in insect antennae and play a critical role in degrading xenobiotics and odorants. In Cydia pomonella, we identified 26 biotransformation enzymes. Among these enzymes, twelve carboxylesterases (CXEs), two aldehyde oxidases (AOXs) and six alcohol dehydrogenases (ADs) were predominantly expressed in antennae. Each of the CpomCXEs presents a conserved catalytic triad "Ser-His-Glu", which is the structural characteristic of known insect CXEs. CpomAOXs present two redox centers, a FAD-binding domain and a molybdenum cofactor/substrate-binding domain. The antennal CpomADs are from two protein families, short-chain dehydrogenases/reducetases (SDRs) and medium-chain dehydrogenases/reducetases (MDRs). Putative catalytic active domain and cofactor binding domain were found in these CpomADs. Potential functions of these enzymes were determined by phylogenetic analysis. The results showed that these enzymes share close relationship with odorant degrading enzymes (ODEs) and resistance-associated enzymes of other insect species. Because of commonly observed roles of insect antennal biotransformation enzymes, we suggest antennal biotransformation enzymes presented here are candidate that involved in degradation of odorants and xenobiotics within antennae of C. pomonella.

  18. Role of farnesoid X receptor in establishment of ontogeny of phase-I drug metabolizing enzyme genes in mouse liver.

    Science.gov (United States)

    Peng, Lai; Piekos, Stephanie; Guo, Grace L; Zhong, Xiao-Bo

    2016-09-01

    The expression of phase-I drug metabolizing enzymes in liver changes dramatically during postnatal liver maturation. Farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis in liver. However, the role of FXR in regulating ontogeny of phase-I drug metabolizing genes is not clear. Hence, we applied RNA-sequencing to quantify the developmental expression of phase-I genes in both Fxr-null and control (C57BL/6) mouse livers during development. Liver samples of male C57BL/6 and Fxr-null mice at 6 different ages from prenatal to adult were used. The Fxr-null showed an overall effect to diminish the "day-1 surge" of phase-I gene expression, including cytochrome P450s at neonatal ages. Among the 185 phase-I genes from 12 different families, 136 were expressed, and differential expression during development occurred in genes from all 12 phase-I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), and epoxide hydrolase (Ephx); reduction: aldoketo reductase (Akr), quinone oxidoreductase (Nqo), and dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). The data also suggested new phase-I genes potentially targeted by FXR. These results revealed an important role of FXR in regulation of ontogeny of phase-I genes.

  19. Involvement of metabolic resistance and F1534C kdr mutation in the pyrethroid resistance mechanisms of Aedes aegypti in India.

    Science.gov (United States)

    Muthusamy, R; Shivakumar, M S

    2015-08-01

    Pesticide resistance poses a serious problem for worldwide mosquito control programs. Resistance to insecticides can be caused by an increased metabolic detoxification of the insecticide and/or by target site insensitivity. In the present study, we estimated the tolerance of Indian Aedes aegypti populations using adult bioassays that revealed high resistance levels of the field populations to permethrin (RR-6, 5.8 and 5.1 folds) compared to our susceptible population. Enzymatic assays revealed increased activities of glutathione S-transferase and carboxylesterase enzymes in the field populations comparatively to the susceptible population. PBO synergist assays did not confirm that cytochrome P450 monooxygenase metabolic detoxification acted as a major cause of resistance. Hence the role of target site resistance was therefore investigated. A single substitution Phe1534Cys in the voltage gated sodium channel was found in domain III, segment 6 (III-S6) of the resistance populations (allele frequency=0.59, 0.51 and 0.47) suggesting its potential role in permethrin resistance in A. aegypti.

  20. Vinyl acetate induces intracellular acidification in mouse oral buccal epithelial cells.

    Science.gov (United States)

    Nakamoto, Tetsuji; Wagner, Mark; Melvin, James E; Bogdanffy, Matthew S

    2005-08-14

    Vinyl acetate exposure in drinking water has been associated with tumor formation in the upper gastrointestinal tract of rats and mice. One potential mechanism for inducing carcinogenesis involves acidification of the intracellular environment due to the metabolism of vinyl acetate to acetic acid. Prolonged intracellular acidification is thought to produce cytotoxic and/or mitogenic responses that are the sentinel pharmacodynamic steps toward cancer. To determine whether exposure to vinyl acetate affects the intracellular pH of intact oral cavity tissue, isolated mouse oral buccal epithelium was loaded with the pH-sensitive dye BCECF, and then exposed to vinyl acetate concentrations ranging from 10 to 1000 microM for up to 4 min. Extracellular vinyl acetate exposure induced a progressive intracellular acidification that was reversible upon removal of the vinyl acetate. The rate of the acidification was concentration-dependent and increased exponentially within the concentration range tested. The magnitude of the vinyl acetate-induced acidification was inhibited by pretreatment with the carboxylesterase inhibitor bis(p-nitrophenyl)phosphate. These results are consistent with the hypothesis that vinyl acetate contributes to the generation and progression of oral cavity tumors via a process of intracellular acidification. Such a process has been proposed to have practical dose-response thresholds below which the intracellular environment can be maintained within homeostatic bounds and the contribution of exposure to carcinogenic risk is negligible.

  1. Candidate chemosensory genes in the Stemborer Sesamia nonagrioides.

    Science.gov (United States)

    Glaser, Nicolas; Gallot, Aurore; Legeai, Fabrice; Montagné, Nicolas; Poivet, Erwan; Harry, Myriam; Calatayud, Paul-André; Jacquin-Joly, Emmanuelle

    2013-01-01

    The stemborer Sesamia nonagrioides is an important pest of maize in the Mediterranean Basin. Like other moths, this noctuid uses its chemosensory system to efficiently interact with its environment. However, very little is known on the molecular mechanisms that underlie chemosensation in this species. Here, we used next-generation sequencing (454 and Illumina) on different tissues from adult and larvae, including chemosensory organs and female ovipositors, to describe the chemosensory transcriptome of S. nonagrioides and identify key molecular components of the pheromone production and detection systems. We identified a total of 68 candidate chemosensory genes in this species, including 31 candidate binding-proteins and 23 chemosensory receptors. In particular, we retrieved the three co-receptors Orco, IR25a and IR8a necessary for chemosensory receptor functioning. Focusing on the pheromonal communication system, we identified a new pheromone-binding protein in this species, four candidate pheromone receptors and 12 carboxylesterases as candidate acetate degrading enzymes. In addition, we identified enzymes putatively involved in S. nonagrioides pheromone biosynthesis, including a ∆11-desaturase and different acetyltransferases and reductases. RNAseq analyses and RT-PCR were combined to profile gene expression in different tissues. This study constitutes the first large scale description of chemosensory genes in S. nonagrioides.

  2. Enzymatic Alterations and Genotoxic Effects Produced by Sublethal Concentrations of Organophosphorous Temephos in Poecilia reticulata.

    Science.gov (United States)

    Pereira, Boscolli Barbosa; de Campos Júnior, Edimar Olegário

    2015-01-01

    The responses of biochemical and genetic parameters were evaluated in tissues of Poecilia reticulata exposed to sublethal and environmentally relevant concentrations of 0.005, 0.01, or 0.02 mg/L of the organophosphorous (OP) pesticide temephos (TE) for 168 h. Activities of enzymes brain acetylcholinesterase (AChE) and liver carboxylesterase (CbE) were determined. Nuclear abnormalities (NA) and micronucleus (MN) frequency in gill erythrocytes were also measured. No mortality was observed over the experimental period; however, brain AChE activities were decreased significantly in guppies in all TE treatment groups after 72 h of exposure. Hepatic CbE activities of fish were increased in all TE treatment groups at 96, 120, and 144 h of exposure. The frequencies of MN and NA in fish gill erythrocytes displayed a marked rise after 168 h of exposure to concentrations of 0.01 or 0.02 mg/L TE. Thus, determination of these parameters may be employed as potential indices of exposure to TE using this sentinel organism for monitorining.

  3. Bioefficacy of Alpinia galanga (Zingiberaceae) rhizome extracts, (E)-p-acetoxycinnamyl alcohol, and (E)-p-coumaryl alcohol ethyl ether against Bactrocera dorsalis (Diptera: Tephritidae) and the impact on detoxification enzyme activities.

    Science.gov (United States)

    Sukhirun, N; Pluempanupat, W; Bullangpoti, V; Koul, O

    2011-10-01

    The application of insecticides to control oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae), is a principal component of the current management of these fruit flies. However, we evaluated four extracts of Alpinia galanga Wild Linn (Zingiberaceae) rhizomes against adult flies and found hexane and ethanol extracts to be most effective (LC50 = 4,866 and 6,337 ppm, respectively, after 24 h). This suggested that both nonpolar and polar compounds could be active in the candidate plant. Accordingly, the hexane extract was further processed to isolate nonpolar active compounds from this plant source. Two compounds, (E)-p-acetoxycinnamyl alcohol and (E)-p-coumaryl alcohol ethyl ether, were identified as active ingredients and found to be more active than total hexane extract (LC50 = 3,654 and 4,044 ppm, respectively, after 24 h). The data suggested that the compounds were not synergistic but may have some additive effect in a mixture. The activity of the hexane extract against detoxification enzymes, carboxylesterase (CE) and glutathione transferase (GST) also was determined in vitro. CE was inhibited by 70%, whereas GST was not significantly inhibited. Insect CEs mediate insecticide resistance via their induction; therefore, inhibition of these enzymes by plant allelochemicals could be a useful alternative approach for the management of the pest in the field.

  4. Toxicology and Biodistribution Studies for MGH2.1, an Oncolytic Virus that Expresses Two Prodrug-activating Genes, in Combination with Prodrugs.

    Science.gov (United States)

    Kasai, Kazue; Nakashima, Hiroshi; Liu, Fang; Kerr, Samantha; Wang, Jiang; Phelps, Mitch; Potter, Philip M; Goins, William B; Fernandez, Soledad A; Chiocca, E Antonio

    2013-08-06

    MGH2.1 is a herpes simplex virus type 1 (HSV1) oncolytic virus that expresses two prodrug-activating transgenes: the cyclophosphamide (CPA)-activating cytochrome P4502B1 (CYP2B1) and the CPT11-activating secreted human intestinal carboxylesterase (shiCE). Toxicology and biodistribution of MGH2.1 in the presence/absence of prodrugs was evaluated in mice. MGH2.1 ± prodrugs was cytotoxic to human glioma cells, but not to normal cells. Pharmacokinetically, intracranial MGH2.1 did not significantly alter the metabolism of intraperitoneally (i.p.) administered prodrugs in mouse plasma, brain, or liver. MGH2.1 did not induce an acute inflammatory reaction. MGH2.1 DNA was detected in brains of mice inoculated with 10(8) pfus for up to 60 days. However, only one animal showed evidence of viral gene expression at this time. Expression of virally encoded genes was restricted to brain. Intracranial inoculation of MGH2.1 did not induce lethality at 10(8) pfus in the absence of prodrugs and at 10(6) pfus in the presence of prodrugs. This study provides safety and toxicology data justifying a possible clinical trial of intratumoral injection of MGH2.1 with peripheral administration of CPA and/or CPT11 prodrugs in humans with malignant gliomas.Molecular Therapy-Nucleic Acids (2013) 2, e113; doi:10.1038/mtna.2013.38; published online 6 August 2013.

  5. Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome

    Directory of Open Access Journals (Sweden)

    Liu Yu

    2008-12-01

    Full Text Available Abstract Background Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes and developing various biotechnological applications. Results The pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45–49%. The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3

  6. Toxicity of L1-2 fraction of Arctium lappa against Panonychus citri (McGregor) (Acari :Tetranychidae) and its effects on several metabolic enzymes%牛蒡L1-2组分对桔全爪螨的毒性和几种代谢酶的作用

    Institute of Scientific and Technical Information of China (English)

    胡军华; 马丽娜; 冉春; 李鸿筠; 姚廷山; 刘浩强; 雷慧德

    2010-01-01

    [目的]探讨杀螨植物牛蒡Arctium lappa L.提取物中主要杀螨成分L1-2的杀螨作用机理.[方法]采用叶片浸渍法处理桔全爪螨Panonychus citri雌成螨后,测定了静止期、兴奋期、痉挛期、麻痹期、复苏和死亡期5个中毒阶段试虫体内几种代谢酶的活性.[结果]L1-2组分在静止期和复苏期对羧酸酯酶(carboxylesterase,CarE)具有一定的抑制作用,在其他时期均激活CarE活性.除了静止期外,在其他时期均能激活乙酰胆碱酯酶(acetylcholinesterase,AChE)和谷胱甘肽转移酶(glutathione S-transferases,GSTs)的活性,在痉挛期和麻痹期活性增强,随后在麻痹期和复苏期降低.[结论]L1-2组分对CarE的抑制与其毒杀活性有关,而中毒试虫的复苏可能与AChE和GSTs有关.该组分可在较长时期内影响桔全爪螨的神经传导及消化和生殖系统,具有潜在的应用研究价值.

  7. Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

    Directory of Open Access Journals (Sweden)

    Richardson Annette C

    2008-07-01

    Full Text Available Abstract Background Kiwifruit (Actinidia spp. are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs. Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons. Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases and pathways (terpenoid biosynthesis is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

  8. Hormone-dependence of sarin lethality in rats: Sex differences and stage of the estrous cycle

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Carl D., E-mail: carl.d.smith179.mil@mail.mil; Wright, Linnzi K.M.; Garcia, Gregory E.; Lee, Robyn B.; Lumley, Lucille A.

    2015-09-15

    Chemical warfare nerve agents (CWNAs) are highly toxic compounds that cause a cascade of symptoms and death, if exposed casualties are left untreated. Numerous rodent models have investigated the toxicity and mechanisms of toxicity of CWNAs, but most are limited to male subjects. Given the profound physiological effects of circulating gonadal hormones in female rodents, it is possible that the daily cyclical fluctuations of these hormones affect females' sensitivity to the lethal effects of CWNAs, and previous reports that included female subjects did not control for the stage of the hormonal cycle. The aim of the current study was to determine the 24-hour median lethal dose (LD{sub 50}) of the CWNA sarin in male, ovariectomized (OVEX) female, and female rats during different stages of the estrous cycle (diestrus, proestrus, and estrus). Additionally, baseline activity levels of plasma acetylcholinesterase, butyrylcholinesterase, and carboxylesterase were measured to determine differences among the groups. Results indicated that females in proestrus had a significantly higher LD{sub 50} of sarin compared to OVEX and estrous females. Although some sex differences were observed in the activity levels of plasma esterases, they were not consistent and likely not large enough to significantly affect the LD{sub 50}s. These results suggest that hormonal cyclicity can influence the outcome of CWNA-related studies using female rodents, and that this variability can be minimized by controlling for the stage of the cycle. Additional research is necessary to determine the precise mechanism of the observed differences because it is unlikely to be solely explained by plasma esterase activity. - Highlights: • The LD{sub 50} of sarin was determined in female rats throughout the stages of the estrous cycle. • Females in proestrus had a significantly higher LD{sub 50} compared to estrous or ovariectomized females. • No sex differences were observed between male and female

  9. Hormone-dependence of sarin lethality in rats: sex differences and stage of the estrous cycle

    Science.gov (United States)

    Smith, Carl D.; Wright, Linnzi K.M.; Garcia, Gregory E.; Lee, Robyn B.; Lumley, Lucille A.

    2015-01-01

    Chemical warfare nerve agents (CWNAs) are highly toxic compounds that cause a cascade of symptoms and death, if exposed casualties are left untreated. Numerous rodent models have investigated the toxicity and mechanisms of toxicity of CWNAs, but most are limited to male subjects. Given the profound physiological effects of circulating gonadal hormones in female rodents, it is possible that the daily cyclical fluctuations of these hormones affect females’ sensitivity to the lethal effects of CWNAs, and previous reports that included female subjects did not control for the stage of the hormonal cycle. The aim of the current study was to determine the 24-hour median lethal dose (LD50) of the CWNA sarin in male, ovariectomized (OVEX) female, and female rats during different stages of the estrous cycle (diestrus, proestrus, and estrus). Additionally, baseline activity levels of plasma acetylcholinesterase, butyrylcholinesterase, and carboxylesterase were measured to determine differences among the groups. Results indicated that females in proestrus had a significantly higher LD50 of sarin compared to OVEX and estrous females. Although some sex differences were observed in the activity levels of plasma esterases, they were not consistent and likely not large enough to significantly affect the LD50s. These results suggest that hormonal cyclicity can influence the outcome of CWNA-related studies using female rodents, and that this variability can be minimized by controlling for the stage of the cycle. Additional research is necessary to determine the precise mechanism of the observed differences because it is unlikely to be solely explained by plasma esterase activity. PMID:26079828

  10. Cholinesterase activity in gilthead seabream (Sparus aurata) larvae: Characterization and sensitivity to the organophosphate azinphosmethyl.

    Science.gov (United States)

    Arufe, M Isabel; Arellano, Juana M; García, Leticia; Albendín, Gemma; Sarasquete, Carmen

    2007-10-15

    Assessment of cholinesterase (ChE) inhibition is widely used as a specific biomarker for evaluating the exposure and effects of non-target organisms to anticholinesterase agents. Cholinesterase and carboxylesterase activities have been measured in larvae of gilthead seabream, Sparus aurata, during the endogenous feeding stage, and ChE was characterized with the aid of diagnostic substrates and inhibitors. The results of the present study showed that whole-body ChE of yolk-sac seabream larvae possesses typical properties of acetylcholinesterase (AChE) with a apparent affinity constant (K(m)) of 0.163+/-0.008 mM and a maximum velocity (V(max)) of 332.7+/-2.8 nmol/min/mg protein. Moreover, sensibility of this enzyme was investigated using the organophosphorus insecticide azinphosmethyl. Static-renewal toxicity tests were conducted over 72 h and larval survival and AChE inhibition were recorded. Mean mortality of seabream larvae increased with increasing concentrations of azinphosmethyl and exposure duration. The estimated 72-h LC50 value to azinphosmethyl was 4.59 microg/l (95% CI=0.46-8.71 microg/l) and inhibition of ChE activity gave an IC50 of 3.04 microg/l (95% CI=2.73-3.31 microg/l). Larvae exposed to azinphosmethyl for 72h showed a 70% inhibition of the whole-body acetylcholinesterase activity at approximately the LC50. In conclusion, the results of the present study suggested that monitoring ChE activity is a valuable tool indicating OP exposure in S. aurata larvae and that acetylthiocholine is the most appropriate substrate for assessing ChE inhibition in this early-life stage of the fish.

  11. A novel family VII esterase with industrial potential from compost metagenomic library

    Directory of Open Access Journals (Sweden)

    Oh Tae-Kwang

    2011-05-01

    Full Text Available Abstract Background Among the vast microbial genomic resources now available, most microbes are unculturable in the laboratory. A culture-independent metagenomic approach is a novel technique that circumvents this culture limitation. For the screening of novel lipolytic enzymes, a metagenomic library was constructed from compost, and the clone of estCS2 was selected for lipolytic properties on a tributyrin-containing medium. Results The estCS2 sequence encodes a protein of 570 amino acid residues, with a predicted molecular mass of 63 kDa, and based on amino acid identity it most closely matches (45% the carboxylesterase from Haliangium ochraceum DSM 14365. EstCS2 belong to family VII, according to the lipolytic enzyme classification proposed by Arpigny and Jaeger, and it retains the catalytic triad Ser245-Glu363-His466 that is typical of an α/β hydrolase. The Ser245 residue in the catalytic triad of EstCS2 is located in the consensus active site motif GXSXG. The EstCS2 exhibits strong activity toward p-nitrophenyl caproate (C6, and it is stable up to 60°C with an optimal enzymatic activity at 55°C. The maximal activity is observed at pH 9, and it remains active between pH 6-10. EstCS2 shows remarkable stability in up to 50% (v/v dimethyl sulfoxide (DMSO or dimethylformamide (DMF. The enzyme has the ability to cleave sterically hindered esters of tertiary alcohol, as well as to degrade polyurethanes, which are widely used in various industries. Conclusions The high stability of EstCS2 in organic solvents and its activity towards esters of ketoprofen and tertiary alcohols, and in polyurethane suggests that it has potential uses for many applications in biotransformation and bioremediation.

  12. Effects of selected xenobiotics on hepatic and plasmatic biomarkers in juveniles of Solea senegalensis.

    Science.gov (United States)

    Solé, Montserrat; Fortuny, Anna; Mañanós, Evaristo

    2014-11-01

    In recent years, Solea senegalensis has increasingly been used in pollution monitoring studies. In order to assess its response to some particular widespread pollutants, juveniles of S. senegalensis were administered an intraperitoneal injection of the model aryl hydrocarbon receptor agonist β-naphtoflavone (βNF) and chemicals of environmental concern, such as the fungicide ketoconazole (KETO), the lipid regulator gemfibrozil (GEM), the surfactant nonylphenol (NP) and the synthetic hormone ethinylestradiol (EE2). Two days after injection, the effect of these chemicals was followed up as alterations of hepatic microsomal activities of the cytochrome P450 (CYPs) and associated reductases, carboxylesterases (CbEs) and the conjugation enzyme uridine diphosphate glucuronyltransferase (UDPGT). In the cytosolic fraction of the liver, the effect on CbEs, glutathione S-transferase (GST) and antioxidant activities was also considered. Alterations on the endocrine reproductive system were evaluated by plasma levels of vitellogenin (VTG) and the sex steroids estradiol (E2), testosterone (T), 11-ketotestosterone (11KT) and the progestin 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). Injection with the model compound βNF induced the hydrolysis rate of the seven CYP substrates assayed. The xenobiotic GEM induced three CYP-related activities (e.g. ECOD) and UDPGT, but depressed antioxidant defenses. EE2 induced four CYPs, more significantly ECOD and BFCOD activities. The xenoestrogens NP and EE2 altered the activities of CbE in microsomes and catalase, and were the only treatments that induced de novo VTG synthesis. In addition, the progestin 17,20β-P, was induced in NP-injected fish. None of the treatments caused statistically significant effects on steroid plasma levels. In conclusion, the CYP substrates assayed responded specifically to treatments and juveniles of S. senegalensis appear good candidates for assessing xenobiotics exposure.

  13. Physiological response of alligator gar juveniles (Atractosteus spatula) exposed to sub-lethal doses of pollutants.

    Science.gov (United States)

    González, Carlos Aguilera; Cruz, Julio; Alfaro, Roberto Mendoza

    2015-08-01

    Alligator gar populations have declined because of overfishing, habitat loss and pollution. Over time, the exposure to different pollutants have affected these fishes as a consequence of their high trophic level, bottom-dwelling habits and long life span. In order to evaluate the physiological effects of pollutants on alligator gar, juveniles (6, 12 and 24 months) were exposed to sub-lethal doses of diazinon, β-naphthoflavone (BNF) and 17 β-estradiol (E2) by intraperitoneal injection. After 2 days of exposure, liver samples were taken to determine the activities of acetylcholinesterase, butyrylcholinesterase and carboxylesterase; alkaline and acid phosphatases (ALP and ACP); ethoxyresorufin o-deethylase (EROD); glutathione s-transferase (GST); superoxide dismutase (SOD), and vitellogenin (VTG) concentration. Two additional bioassays consisting on the exposure of compounds through water or food were performed and after 4 and 28 days, respectively, biomarkers were determined. All esterases were inhibited in organisms exposed to diazinon as well as in 6-months gar exposed to E2 and BNF. In contrast, ALP activity increased in gar exposed to diazinon and E2, while ACP activity did not show any variations. No EROD activity was registered after exposure to the different pollutants, despite being one of the most sensitive and common detoxification biomarkers used for fishes. GST activity reduction was detected when gar were exposed to E2 and BNF, while SOD activity increased after exposure to diazinon and E2. Finally, VTG levels were higher in animals exposed to E2 compared to other treatments. Overall, these results suggest that alligator gar juveniles have a low biotransformation metabolism and show that they are especially sensitive to those pollutants affecting the nervous system.

  14. Insecticide resistance in the sand fly, Phlebotomus papatasi from Khartoum State, Sudan

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    Hassan Mo'awia

    2012-03-01

    Full Text Available Abstract Background Phlebotomus papatasi the vector of cutaneous leishmaniasis (CL is the most widely spread sand fly in Sudan. No data has previously been collected on insecticide susceptibility and/or resistance of this vector, and a first study to establish a baseline data is reported here. Methods Sand flies were collected from Surogia village, (Khartoum State, Rahad Game Reserve (eastern Sudan and White Nile area (Central Sudan using light traps. Sand flies were reared in the Tropical Medicine Research Institute laboratory. The insecticide susceptibility status of first progeny (F1 of P. papatasi of each population was tested using WHO insecticide kits. Also, P. papatasi specimens from Surogia village and Rahad Game Reserve were assayed for activities of enzyme systems involved in insecticide resistance (acetylcholinesterase (AChE, non-specific carboxylesterases (EST, glutathione-S-transferases (GSTs and cytochrome p450 monooxygenases (Cyt p450. Results Populations of P. papatasi from White Nile and Rahad Game Reserve were sensitive to dichlorodiphenyltrichloroethane (DDT, permethrin, malathion, and propoxur. However, the P. papatasi population from Surogia village was sensitive to DDT and permethrin but highly resistant to malathion and propoxur. Furthermore, P. papatasi of Surogia village had significantly higher insecticide detoxification enzyme activity than of those of Rahad Game Reserve. The sand fly population in Surogia displayed high AChE activity and only three specimens had elevated levels for EST and GST. Conclusions The study provided evidence for malathion and propoxur resistance in the sand fly population of Surogia village, which probably resulted from anti-malarial control activities carried out in the area during the past 50 years.

  15. Intervention effect of traditional Chinese medicine Yi Tang Kang on metabolic syndrome of spleen deficiency

    Institute of Scientific and Technical Information of China (English)

    Xiao-Xi Liu; Yan Shi

    2015-01-01

    Objective:To investigate effects of herbal compoundYiTangKang on the spleen deficiency metabolic syndrome.Methods:Forty maleWistar rats were randomly divided into two groups: the normal control group and theMS spleen deficiency syndrome group.The control group rats were fed with standard diet and water, whileMS spleen deficiency syndrome group with high fat diet and low dose intraperitoneal injection ofstreptozocin, which swam to the endurance limit. After12 weeks, theMS spleen deficiency syndrome group was randomly divided into two groups, with13 rats in each group.Rats in model group were fed with high fat diet and continuouly administered with daily saline, and rats in intervention group with high fat diet were trated with traditionalChinese medicinesYiTangKang by gavage,2 mL/200 g at the same time every day.10 weeks later, the expression of serum proteomics was investigated through abdominal aortic puncture and separation of serum, using isotope labeling technique, high performance liquid chromatography and four bar-Orbitrap mass spectrometer.Results:After treatment with traditionalChinese medicine yitangkang, in the model group, important carboxylesterase and retinal guanylatecyclase2 precursor were upregulated.As for intervention group, these indesxes were raised, but immunoglobulinIgG, carnitine acetyltransferase, tubulin beta -5, andGanLu sugar binding proteinC were down-regulated.At the same time, some new biological active substances, such as protein tyrosine kinase, beta glucosidase were also found.Conclusions:TraditionalChinese medicinesYiTangKang could regulate glucose and lipid metabolism in rats with spleen deficiency syndrome.

  16. Genetic diversity, acaricide resistance status and evolutionary potential of a Rhipicephalus microplus population from a disease-controlled cattle farming area in South Africa.

    Science.gov (United States)

    Robbertse, Luïse; Baron, Samantha; van der Merwe, Nicolaas A; Madder, Maxime; Stoltsz, Wilhelm H; Maritz-Olivier, Christine

    2016-06-01

    The Southern cattle tick, Rhipicephalus microplus is a hematophagous ectoparasite of great veterinary and economic importance. Along with its adaptability, reproductive success and vectoring capacity, R. microplus has been reported to develop resistance to the major chemical classes of acaricides currently in use. In South Africa, the Mnisi community in the Mpumalanga region offers a unique opportunity to study the adaptive potential of R. microplus. The aims of this study therefore included characterising acaricide resistance and determining the level and pattern of genetic diversity for R. microplus in this region from one primary population consisting of 12 communal dip-stations. The level of acaricide resistance was evaluated using single nucleotide polymorphisms (SNPs) in genes that contribute to acaricide insensitivity. Additionally, the ribosomal internal transcribed spacer 2 (ITS2) gene fragments of collected individuals were sequenced and a haplotype network was constructed. A high prevalence of alleles attributed to resistance against formamidines (amitraz) in the octopamine/tyramine (OCT/Tyr) receptor (frequency of 0.55) and pyrethroids in the carboxylesterase (frequency of 0.81) genes were observed. Overall, the sampled tick population was homozygous resistant to pyrethroid-based acaricides in the voltage-gated sodium channel (VGS) gene. A total of 11 haplotypes were identified in the Mnisi R. microplus population from ITS2 analysis with no clear population structure. From these allele frequencies it appears that formamidine resistance in the Mnisi community is on the rise, as the R. microplus populations is acquiring or generating these resistance alleles. Apart from rearing multi-resistant ticks to commonly used acaricides in this community these ticks may pose future problems to its surrounding areas.

  17. Physiological and biochemical effects of botanical extract from Piper nigrum Linn (Piperaceae) against the dengue vector Aedes aegypti Liston (Diptera: Culicidae).

    Science.gov (United States)

    Lija-Escaline, Jalasteen; Senthil-Nathan, Sengottayan; Thanigaivel, Annamalai; Pradeepa, Venkatraman; Vasantha-Srinivasan, Prabhakaran; Ponsankar, Athirstam; Edwin, Edward Sam; Selin-Rani, Selvaraj; Abdel-Megeed, Ahmed

    2015-11-01

    The leaves of Piper nigrum L. (Piperaceae) were evaluated for chemical constituents and mosquito larvicidal activity against the larvae of Aedes aegypti. GC and GC-MS analyses revealed that the crude extracts contain 16 compounds. Thymol (20.77%) and ç-elemene (10.42%) were identified as the major constituents followed by cyclohexene, 4-ethenyl-4-methyl-3-(1-methylethenyl)-1-(1 methylethyl)-, (3R-trans) (7.58%), 4,6-octadienoic acid, 2-acetyl-2-methyl-, ethyl ester (6.98), 2(3H)-furanone, 3,4-bis(1,3-benzodioxol-5-ylmethyl) dihydro-, (3R-trans) (6.95%), 1-naphthalenol, 1,2,3,4,4a,7,8,8a-octahydro-1,6-dimethyl-4-(1-methylethyl)-, [1R-(1à,4á,4aá,8aá)]-(Cedreanol) (5.30%), trans-2-undecen-1-ol (4.48%), phytol (4.22%), 1,6-cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-,[s-(E,E)] (3.78%) and 2,6-dimethyl-3,5,7-octatriene-2-ol, Z,Z (2.39%). Larval mortality was observed after 3 h of exposure period. The crude extract showed remarkable larvicidal activity against Ae. aegypti (LC50 = 34.97). The larvae of Ae. aegypti exposed to the P. nigrum, significantly reduced the activities of α- and β-carboxylesterases and superdioxide. Further, P. nigrum extract was severely affecting the mosquito gut cellular organelles. Based on the results, the chemical constituents of crude extracts of P. nigrum can be considered as a new source of larvicide for the control of Ae. aegypti.

  18. Comparative analysis of mosquito (Diptera: Culicidae: Aedes aegypti Liston) responses to the insecticide Temephos and plant derived essential oil derived from Piper betle L.

    Science.gov (United States)

    Vasantha-Srinivasan, Prabhakaran; Senthil-Nathan, Sengottayan; Ponsankar, Athirstam; Thanigaivel, Annamalai; Edwin, Edward-Sam; Selin-Rani, Selvaraj; Chellappandian, Muthiah; Pradeepa, Venkatraman; Lija-Escaline, Jalasteen; Kalaivani, Kandaswamy; Hunter, Wayne B; Duraipandiyan, Veeramuthu; Al-Dhabi, Naif Abdullah

    2017-05-01

    Resistance to treatments with Temephos or plant derived oil, Pb-CVO, between a field collected Wild Strain (WS) and a susceptible Laboratory Strain (LS) of Ae. aegypti were measured. The Temephos (0.1mg/L) showed the greatest percentage of mosquito mortality compared to Pb-CVO (1.5mg/L) in LS Ae. aegypti. However, WS Ae. aegypti was not significantly affected by Temephos (0.1mg/L) treatment compare to the Pb-CVO (1.5mg/L). However, both strains (LS and WS) when treated with Pb-CVO (1.5mg/L) displayed steady larval mortality rate across all instars. The LC50 of Temephos was 0.027mg in LS, but increased in WS to 0.081mg/L. The LC50 of Pb-CVO treatment was observed at concentrations of 0.72 and 0.64mg/L for LS and WS strains respectively. The enzyme level of α- and β-carboxylesterase was reduced significantly in both mosquito strains treated with Pb-CVO. Whereas, there was a prominent deviation in the enzyme ratio observed between LS and WS treated with Temephos. The GST and CYP450 levels were upregulated in the LS, but decreased in WS, after treatment with Temephos. However, treatment with Pb-CVO caused both enzyme levels to increase significantly in both the strains. Visual observations of the midgut revealed cytotoxicity from sub-lethal concentrations of Temephos (0.04mg/L) and Pb-CVO (1.0mg/L) in both strains of Ae. aegypti compared to the control. The damage caused by Temephos was slightly less in WS compared to LS mosquito strains.

  19. Chemical composition and insecticidal property of Myrsine stolonifera (Koidz.) walker (Family: Myrsinaceae) on Musca domestica (Diptera: Muscidae).

    Science.gov (United States)

    Wang, Xue Gui; Li, Qian; Jiang, Su Rong; Li, Pei; Yang, Ji Zhi

    2017-02-22

    Musca domestica is one of the most important pests of human health, and has developed strong resistance to many chemicals used for its control. One important approach for creating new pesticides is the exploration of novel compounds from plants. During a wide screening of plants with insecticidal properties that grow in southern China, we found that the methanolic extracts of Myrsine stolonifera had insecticidal activity against the adults of M. domestica. However, the insecticidal constituents and mechanisms of the M. stolonifera extracts remain unclear. The insecticidal components of the methanolic extracts of M. stolonifera were isolated with activity-guided fractionation. From the spectra of nuclear magnetic resonance (NMR) and mass spectrometry (MS), the compounds were identified as syringing (1), 2,6-dimethoxy-4-hydroxyphenol-1-O-β-d-glu (2), kaempferol-3-O-glu-rha-glu (3), and quercetin-3-O-glu-rha-glu (4). This study is the first to report the spectral data for compounds 3 and 4, and their LC50 values were 0.52mg/g sugar and 0.36mg/g sugar 24h after treatment of the adults of M. domestica, respectively. Compounds 3 and 4 (LC25) also inhibited the activities of the enzymes carboxylesterase, glutathione S-transferase, mixed function oxidase, and acetylcholine esterase of adult M. domestica, particularly mixed function oxidase and acetylcholine esterase. The cytotoxic effects of compounds 3 and 4 on cell proliferation, mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) were demonstrated on SL-1 cells. From the extracts of M. stolonifera, quercetin-3-O-glu-rha-glu and kaempferol-3-O-glu-rha-glu have displayed comparable toxicities to rotenone on M. domestica and also exhibited cytotoxic effects on SL-1 cells; therefore, the extracts of M. stolonifera and their compounds have potential as botanical insecticides to control M. domestica.

  20. Degeneration and recovery of rat olfactory epithelium following inhalation of dibasic esters.

    Science.gov (United States)

    Keenan, C M; Kelly, D P; Bogdanffy, M S

    1990-08-01

    Dibasic esters (DBE) are solvent mixtures used in the paint and coating industry. To evaluate the potential subchronic toxicity of DBE, groups of male and female rats were exposed for periods of up to 13 weeks to DBE concentrations of 0, 20, 76, or 390 mg/m3. After approximately 7 and 13 weeks of exposure, 10 rats per sex per group were subjected to clinical chemical, hematological, and urine analyses. Following 7 or 13 weeks of exposure, 10 or 20 rats per sex per group, respectively, were euthanized. An additional 10 rats were euthanized following a 6-week recovery period. A standard profile of tissues, including four levels of nasal cavity, was evaluated histopathologically. After 7 weeks of exposure, slight degeneration of the olfactory epithelium was observed in both male and female rats at 76 and 390 mg/m3. After 13 weeks, degeneration of the olfactory epithelium was present at all DBE concentrations in female rats, but only at the mid and high concentrations in male rats. The severity and incidence of the lesions were concentration related for both sexes with female rats being more sensitive than males. Following the recovery period, histological changes compatible with repair in the olfactory mucosa included an absence of degeneration, focal disorganization of the olfactory epithelium, and respiratory metaplasia. All other tissues were macroscopically normal. No other signs of toxicity were indicated by the other parameters evaluated. Inhalation studies of other esters demonstrate similar pathology in the olfactory epithelium. Since olfactory mucosa is rich in carboxylesterase activity, acids may be the toxic metabolites of these compounds. This hypothetical mechanism may explain the sensitivity of olfactory tissue to the effects of DBE.

  1. Repeated low-dose exposures to sarin, soman, or VX affect acoustic startle in guinea pigs.

    Science.gov (United States)

    Smith, C D; Lee, R B; Moran, A V; Sipos, M L

    2016-01-01

    Chemical warfare nerve agents (CWNAs) are known to cause behavioral abnormalities in cases of human exposures and in animal models. The behavioral consequences of single exposures to CWNAs that cause observable toxic signs are particularly well characterized in animals; however, less is known regarding repeated smaller exposures that may or may not cause observable toxic signs. In the current study, guinea pigs were exposed to fractions (0.1, 0.2, or 0.4) of a medial lethal dose (LD50) of sarin, soman, or VX for two weeks. On each exposure day, and for a post-exposure period, acoustic startle response (ASR) was measured in each animal. Although relatively few studies use guinea pigs to measure behavior, this species is ideal for CWNA-related experiments because their levels of carboxylesterases closely mimic those of humans, unlike rats or mice. Results showed that the 0.4 LD50 doses of soman and VX transiently increased peak startle amplitude by the second week of injections, with amplitude returning to baseline by the second week post-exposure. Sarin also increased peak startle amplitude independent of week. Latencies to peak startle and PPI were affected by agent exposure but not consistently among the three agents. Most of the changes in startle responses returned to baseline following the cessation of exposures. These data suggest that doses of CWNAs not known to produce observable toxic signs in guinea pigs can affect behavior in the ASR paradigm. Further, these deficits are transient and usually return to baseline shortly after the end of a two-week exposure period.

  2. Intestinal glucuronidation protects against chemotherapy-induced toxicity by irinotecan (CPT-11).

    Science.gov (United States)

    Chen, Shujuan; Yueh, Mei-Fei; Bigo, Cyril; Barbier, Olivier; Wang, Kepeng; Karin, Michael; Nguyen, Nghia; Tukey, Robert H

    2013-11-19

    Camptothecin (CPT)-11 (irinotecan) has been used widely for cancer treatment, particularly metastatic colorectal cancer. However, up to 40% of treated patients suffer from severe late diarrhea, which prevents CPT-11 dose intensification and efficacy. CPT-11 is a prodrug that is hydrolyzed by hepatic and intestinal carboxylesterase to form SN-38, which in turn is detoxified primarily through UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed glucuronidation. To better understand the mechanism associated with toxicity, we generated tissue-specific Ugt1 locus conditional knockout mouse models and examined the role of glucuronidation in protecting against irinotecan-induced toxicity. We targeted the deletion of the Ugt1 locus and the Ugt1a1 gene specifically in the liver (Ugt1(ΔHep)) and the intestine (Ugt1(ΔGI)). Control (Ugt1(F/F)), Ugt1(ΔHep), and Ugt1(ΔGI) adult male mice were treated with different concentrations of CPT-11 daily for four consecutive days. Toxicities were evaluated with regard to tissue glucuronidation potential. CPT-11-treated Ugt1(ΔHep) mice showed a similar lethality rate to the CPT-11-treated Ugt1(F/F) mice. However, Ugt1(ΔGI) mice were highly susceptible to CPT-11-induced diarrhea, developing severe and lethal mucositis at much lower CPT-11 doses, a result of the proliferative cell loss and inflammation in the intestinal tract. Comparative expression levels of UGT1A1 in intestinal tumors and normal surrounding tissue are dramatically different, providing for the opportunity to improve therapy by differential gene regulation. Intestinal expression of the UGT1A proteins is critical toward the detoxification of SN-38, whereas induction of the UGT1A1 gene may serve to limit toxicity and improve the efficacy associated with CPT-11 treatment.

  3. Thiamethoxam Resistance in the House Fly, Musca domestica L.: Current Status, Resistance Selection, Cross-Resistance Potential and Possible Biochemical Mechanisms.

    Directory of Open Access Journals (Sweden)

    Hafiz Azhar Ali Khan

    Full Text Available The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR at LC50 ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible, the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF and piperonyl butoxide (PBO produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively. In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE and mixed function oxidase (MFO in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.

  4. Thiamethoxam resistance selected in the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae): cross-resistance patterns, possible biochemical mechanisms and fitness costs analysis.

    Science.gov (United States)

    Gao, Cong-Fen; Ma, Shao-Zhi; Shan, Cai-Hui; Wu, Shun-Fan

    2014-09-01

    The western flower thrips (WFT) Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), an important pest of various crops in the world, has invaded China since 2003. To understand the risks and to determine possible mechanisms of resistance to thiamethoxam in WFT, a resistant strain was selected under the laboratory conditions. Cross-resistance and the possible biochemical resistance mechanisms were investigated in this study. A 15.1-fold thiamethoxam-resistant WFT strain (TH-R) was established after selection for 55 generations. Compared with the susceptible strain (TH-S), the selected TH-R strain showed extremely high level cross-resistance to imidaclothiz (392.1-fold) and low level cross-resistance to dinotefuran (5.7-fold), acetamiprid (2.9-fold) and emamectin benzoate (2.1-fold), respectively. No cross-resistance to other fourteen insecticides was detected. Synergism tests showed that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) produced a high synergism of thiamethoxam effects in the TH-R strain (2.6- and 2.6-fold respectively). However, diethyl maleate (DEM) did not act synergistically with thiamethoxam. Biochemical assays showed that mixed function oxidase (MFO) activities and carboxylesterase (CarE) activity of the TH-R strain were 2.8- and 1.5-fold higher than that of the TH-S strain, respectively. When compared with the TH-S strain, the TH-R strain had a relative fitness of 0.64. The results show that WFT develops resistance to thiamethoxam after continuous application and thiamethoxam resistance had considerable fitness costs in the WFT. It appears that enhanced metabolism mediated by cytochrome P450 monooxygenases and CarE was a major mechanism for thiamethoxam resistance in the WFT. The use of cross-resistance insecticides, including imidaclothiz and dinotefuran, should be avoided for sustainable resistance management.

  5. Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: application to the systemic insecticide thiamethoxam.

    Science.gov (United States)

    Badiou-Bénéteau, Alexandra; Carvalho, Stephan M; Brunet, Jean-Luc; Carvalho, Geraldo A; Buleté, Audrey; Giroud, Barbara; Belzunces, Luc P

    2012-08-01

    This study describes the development of acetylcholinesterase (AChE), carboxylesterases (CaE1, CaE2, CaE3), glutathion-S-transferase (GST), alkaline phosphatase (ALP) and catalase (CAT) as enzyme biomarkers of exposure to xenobiotics such as thiamethoxam in the honey bee Apis mellifera. Extraction efficiency, stability under freezing and biological variability were studied. The extraction procedure achieved good recovery rates in one extraction step and ranged from 65 percent (AChE) to 97.3 percent (GST). Most of the enzymes were stable at -20°C, except ALP that displayed a slight but progressive decrease in its activity. Modifications of enzyme activities were considered after exposure to thiamethoxam at the lethal dose 50 percent (LD(50), 51.16 ng bee(-1)) and two sublethal doses, LD(50)/10 (5.12 ng bee(-1)) and LD(50)/20 (2.56 ng bee(-1)). The biomarker responses revealed that, even at the lowest dose used, exposure to thiamethoxam elicited sublethal effects and modified the activity of CaEs, GST, CAT and ALP. Different patterns of biomarker responses were observed: no response for AChE, an increase for GST and CAT, and differential effects for CaEs isoforms with a decrease in CaE1 and CaE3 and an increase in CaE2. ALP and CaE3 displayed contrasting variations but only at 2.56 ng bee(-1). We consider that this profile of biomarker variation could represent a useful fingerprint to characterise exposure to thiamethoxam in the honey bee A. mellifera. This battery of honey bee biomarkers might be a promising option to biomonitor the health of aerial and terrestrial ecosystems and to generate valuable information on the modes of action of pesticides.

  6. Thiamethoxam Resistance in the House Fly, Musca domestica L.: Current Status, Resistance Selection, Cross-Resistance Potential and Possible Biochemical Mechanisms.

    Science.gov (United States)

    Khan, Hafiz Azhar Ali; Akram, Waseem; Iqbal, Javaid; Naeem-Ullah, Unsar

    2015-01-01

    The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC50 ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.

  7. Further assessment of an in vitro screen that may help identify organophosphorus pesticides that are more acutely toxic to the young.

    Science.gov (United States)

    Padilla, S; Sung, H-J; Moser, V C

    2004-09-24

    Some, but not all, organophosphorus pesticides are more acutely toxic to the young as compared to adults. We have developed an in vitro assay that measures the detoxification potential (via carboxylesterase and A-esterases) of tissues. Previous results using this in vitro screen correlated with the marked in vivo sensitivity of the young to chlorpyrifos and also correlated with the equal sensitivity of the young and adult to methamidophos (Padilla et al., 2000). We have now extended these observations to two other pesticides that have already been shown in the literature to be more toxic to the young: parathion (paraoxon) and malathion (malaoxon). In our in vitro assay, liver or plasma from 7-d-old rats were much less efficacious than adult tissues at detoxification of the active metabolites of these two pesticides. Using our in vitro assay we also tested the active metabolite of diazinon, diazoxon, and again found that young liver or plasma possessed much less detoxification capability than adult tissues. From these results, we predicted that young animals would be more sensitive to diazinon, which, in fact, was the case: When postnatal day (PND) 17 or adult rats were given a dosage of 75 mg/kg diazinon, adult brain cholinesterase (ChE) was only inhibited 38%, while the brain ChE in the PND 17 animals showed much more inhibition (75%). We conclude that our in vitro screen may prove to be a useful, quick, convenient test for identifying which organophosphorus pesticides may be more acutely toxic to the young as compared to adults.

  8. An improved chemo-enzymatic synthesis of 1-beta-O-acyl glucuronides: highly chemoselective enzymatic removal of protecting groups from corresponding methyl acetyl derivatives.

    Science.gov (United States)

    Baba, Akiko; Yoshioka, Tadao

    2007-12-07

    An improved and widely applicable chemo-enzymatic method for the synthesis of a series of 1-beta-O-acyl glucuronides 5a-f has been developed from the corresponding methyl acetyl derivatives 3a-f, which were stereospecifically synthesized from cesium salts of carboxylic acids 1a-f and methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate (2). Chemoselectivity of lipase AS Amano (LAS) in the hydrolytic removal of O-acetyl groups of 3a-f to provide methyl esters 4a-f was influenced by the nature of their 1-beta-O-acyl groups; high selectivity was evident only for 3b and 3f. Carboxylesterase from Streptomyces rochei (CSR), newly screened as an alternative to LAS, showed much greater chemoselectivity toward the O-acetyl groups than LAS; 3a, 3d, and 3e were chemoselectively hydrolyzed only by CSR. The combination of CSR with LAS yielded better results in the hydrolysis of 3c and 3f than did single usage of CSR. Final deprotection of the methyl ester groups of 4a-f to provide 5a-f was chemoselectively achieved by using lipase from Candida antarctica type B (CAL-B) as well as esterase from porcine liver (PLE), although CAL-B possessed higher chemoselectivity and catalytic efficiency than did PLE. CSR also exhibited high chemoselectivity in the synthesis of (S)-naproxen 1-beta-O-acyl glucopyranoside (7) from its 2,3,4,6-tetra-O-acetyl derivative 6.

  9. Transcriptomic and Expression Analysis of the Salivary Glands in White-Backed Planthoppers, Sogatella furcifera.

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    Zhen Li

    Full Text Available The white-backed planthopper (WBPH, Sogatella furcifera (Horváth, is one of the serious rice pests because of its destructive feeding. The salivary glands of the WBPH play an important role in the feeding behaviour. Currently, however, very little is known about the salivary glands at the molecular level. We sequenced the salivary gland transcriptome (sialotranscripome of adult WBPHs using the Illumina sequencing. A total of 65,595 transcripts and 51,842 unigenes were obtained from salivary glands. According to annotations against the Nr database, many of the unigenes identified were associated with the most studied enzymes in hemipteran saliva. In the present study, we identified 32 salivary protein genes from the WBPH sialotranscripome, which were categorized as those involved in sugar metabolism, detoxification, suppression of plant defense responses, immunity-related responses, general digestion, and other phytophagy processes. Tissue expression profiles analysis revealed that four of 32 salivary protein genes (multicopper oxidase 4, multicopper oxidase 6, carboxylesterase and uridine phosphorylase 1 isform X2 were primarily expressed in the salivary gland, suggesting that they played putative role in insect-rice interactions. 13 of 32 salivary protein genes were primarily expressed in gut, which might play putative role in digestive and detoxify mechanism. Development expression profiles analysis revealed that the expression level of 26 of 32 salivary protein genes had no significant difference, suggesting that they may play roles in every developmental stages of salivary gland of WBPH. The other six genes have a high expression level in the salivary gland of adult. 31 of 32 genes (except putative acetylcholinesterase 1 have no significant difference in male and female adult, suggesting that their expression level have no difference between sexes. This report analysis of the sialotranscripome for the WBPH, and the transcriptome provides a

  10. Metabolism of ciclesonide in the upper and lower airways: review of available data

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    Ruediger Nave

    2008-09-01

    Full Text Available Ruediger Nave, Nigel McCrackenNycomed GmbH, Konstanz, GermanyAbstract: Ciclesonide is a novel corticosteroid (CS for the treatment of asthma and allergic rhinitis. After administration, the parent compound ciclesonide is converted by intracellular airway esterases to its pharmacologically active metabolite desisobutyryl-ciclesonide (des-CIC. We investigated the in vitro activation of ciclesonide and further esterification of des-CIC to (mainly des-CIC oleate in several human target organ test systems. Human precision-cut lung slices, alveolar type II epithelial cells (A549, normal bronchial epithelial cells (NHBE, and nasal epithelial cells (HNEC were incubated with ciclesonide. Enzymes characterization and the determination of the reversibility of fatty acid esterifi cation was investigated in HNEC and NHBE. Ciclesonide was taken up and converted to des-CIC in all cellular test systems. Intracellular concentrations of des-CIC were maintained for up to 24 h. Formation of des-CIC oleate increased over time in HNEC, A549 cells, and lung slices. The formed des-CIC fatty acid conjugates were reconverted to des-CIC. Increasing concentrations of carboxylesterase and cholinesterase inhibitors progressively reduced the formation of metabolites. The results derived from these studies demonstrate the activation of ciclesonide to des-CIC in the upper and lower airways. The reversible formation of des-CIC fatty acid conjugates may prolong the anti-inflammatory activity of des-CIC and may allow for once-daily dosing.Keywords: ciclesonide, des-CIC, metabolism, human, lung, nasal tissue

  11. Alteration of substrate specificities of thermophilic α/β hydrolases through domain swapping and domain interface optimization

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Zhou; Honglei Wang; Yuhang Zhang; Le Gao; Yan Feng

    2012-01-01

    Protein domain swapping is an efficient way in protein functional evolution in vivo and also has been proved to be an effective strategy to modify the function of the multidomain proteins in vitro.To explore the potentials of domain swapping for alteration of the enzyme substrate specificities and the structure-function relationship of the homologous proteins,here we constructed two chimeras from a pair of thermophilic members of the α/β hydrolase superfamily by grafting their functional domains to the conserved α/β hydrolase fold domain:a carboxylesterase from Archaeoglobus fulgidus (AFEST) and an acylpeptide hydrolase from Aeropyrum pernix K1 (apAPH) and explored their activities on hydrolyze p-nitrophenyl esters (pNP) with different acyl chain lengths.We took two approaches to reduce the crossover disruptions when creating the chimeras:chose the residue which involved in the least contacts as the splicing site and optimized the newly formed domain interfaces of the chimeras by sitedirected mutations.Characterizations of AAM7 and PAR showed that these chimeras inherited the thermophilic property of both parents.In the aspect of substrate specificity,AAM7 and PAR showed highest activity towards short chain length substrate pNPC4 and middle chain length substrate pNPC8,similar to parent AFEST and apAPH,respectively.These results suggested that the substrate-binding domain is the dominant factor on enzyme substrate specificity,and the optimization of the newly formed domain interface is an important guarantee for successful domain swapping of proteins with low-sequence homology.

  12. Involvement of Three Esterase Genes from Panonychus citri (McGregor) in Fenpropathrin Resistance.

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    Shen, Xiao-Min; Liao, Chong-Yu; Lu, Xue-Ping; Wang, Zhe; Wang, Jin-Jun; Dou, Wei

    2016-08-19

    The citrus red mite, Panonychus citri (McGregor), is a major citrus pest with a worldwide distribution and an extensive record of pesticide resistance. However, the underlying molecular mechanism associated with fenpropathrin resistance in this species have not yet been reported. In this study, synergist triphenyl phosphate (TPP) dramatically increased the toxicity of fenpropathrin, suggesting involvement of carboxylesterases (CarEs) in the metabolic detoxification of this insecticide. The subsequent spatiotemporal expression pattern analysis of PcE1, PcE7 and PcE9 showed that three CarEs genes were all over-expressed after insecticide exposure and higher transcripts levels were observed in different field resistant strains of P. citri. Heterologous expression combined with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay in Spodoptera frugiperda (Sf9) cells revealed that PcE1-, PcE7- or PcE9-expressing cells showed significantly higher cytoprotective capability than parental Sf9 cells against fenpropathrin, demonstrating that PcEs probably detoxify fenpropathrin. Moreover, gene silencing through the method of leaf-mediated dsRNA feeding followed by insecticide bioassay increased the mortalities of fenpropathrin-treated mites by 31% (PcE1), 27% (PcE7) and 22% (PcE9), respectively, after individual PcE gene dsRNA treatment. In conclusion, this study provides evidence that PcE1, PcE7 and PcE9 are functional genes mediated in fenpropathrin resistance in P. citri and enrich molecular understanding of CarEs during the resistance development of the mite.

  13. De Novo Assembly and Characterization of the Transcriptome of Grasshopper Shirakiacris shirakii

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    Zhongying Qiu

    2016-07-01

    Full Text Available Background: The grasshopper Shirakiacris shirakii is an important agricultural pest and feeds mainly on gramineous plants, thereby causing economic damage to a wide range of crops. However, genomic information on this species is extremely limited thus far, and transcriptome data relevant to insecticide resistance and pest control are also not available. Methods: The transcriptome of S. shirakii was sequenced using the Illumina HiSeq platform, and we de novo assembled the transcriptome. Results: Its sequencing produced a total of 105,408,878 clean reads, and the de novo assembly revealed 74,657 unigenes with an average length of 680 bp and N50 of 1057 bp. A total of 28,173 unigenes were annotated for the NCBI non-redundant protein sequences (Nr, NCBI non-redundant nucleotide sequences (Nt, a manually-annotated and reviewed protein sequence database (Swiss-Prot, Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG databases. Based on the Nr annotation results, we manually identified 79 unigenes encoding cytochrome P450 monooxygenases (P450s, 36 unigenes encoding carboxylesterases (CarEs and 36 unigenes encoding glutathione S-transferases (GSTs in S. shirakii. Core RNAi components relevant to miroRNA, siRNA and piRNA pathways, including Pasha, Loquacious, Argonaute-1, Argonaute-2, Argonaute-3, Zucchini, Aubergine, enhanced RNAi-1 and Piwi, were expressed in S. shirakii. We also identified five unigenes that were homologous to the Sid-1 gene. In addition, the analysis of differential gene expressions revealed that a total of 19,764 unigenes were up-regulated and 4185 unigenes were down-regulated in larvae. In total, we predicted 7504 simple sequence repeats (SSRs from 74,657 unigenes. Conclusions: The comprehensive de novo transcriptomic data of S. shirakii will offer a series of valuable molecular resources for better studying insecticide resistance, RNAi and molecular marker discovery in the transcriptome.

  14. Multiple insecticide resistance mechanisms involving metabolic changes and insensitive target sites selected in anopheline vectors of malaria in Sri Lanka

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    Karunaratne SHP Parakrama

    2008-08-01

    Full Text Available Abstract Background The current status of insecticide resistance and the underlying resistance mechanisms were studied in the major vector of malaria, Anopheles culicifacies, and the secondary vector, Anopheles subpictus in five districts (Anuradhapura, Kurunegala, Moneragala, Puttalam and Trincomalee of Sri Lanka. Eight other anophelines, Anopheles annularis, Anopheles barbirostris, Anopheles jamesii, Anopheles nigerrimus, Anopheles peditaeniatus, Anopheles tessellatus, Anopheles vagus and Anopheles varuna from Anuradhapura district were also tested. Methods Adult females were exposed to the WHO discriminating dosages of DDT, malathion, fenitrothion, propoxur, λ-cyhalothrin, cyfluthrin, cypermethrin, deltamethrin, permethrin and etofenprox. The presence of metabolic resistance by esterase, glutathione S-transferase (GST and monooxygenase-based mechanisms, and the sensitivity of the acetylcholinesterase target site were assessed using synergists, and biochemical, and metabolic techniques. Results All the anopheline species had high DDT resistance. All An. culicifacies and An. subpictus populations were resistant to malathion, except An. culicifacies from Kurunegala, where there was no malathion carboxylesterase activity. Kurunegala and Puttalam populations of An. culicifacies were susceptible to fenitrothion. All the An. culicifacies populations were susceptible to carbamates. Both species were susceptible to the discriminating dosages of cypermethrin and cyfluthrin, but had different levels of resistance to other pyrethroids. Of the 8 other anophelines, only An. nigerrimus and An. peditaeniatus were resistant to all the insecticides tested, probably due to their high exposure to the insecticides used in agriculture. An. vagus showed some resistance to permethrin. Esterases, GSTs and monooxygenases were elevated in both An. culicifacies and An. subpictus. AChE was most sensitive to insecticides in Kurunegala and Trincomalee An. culicifacies

  15. 蚊虫抗药性分子机制研究进展%Advances in the molecular mechanisms of mosquito resistance to insecticides

    Institute of Scientific and Technical Information of China (English)

    刘宏美; 代玉华; 公茂庆

    2012-01-01

    蚊虫是重要的医学昆虫,可以通过叮咬传播疾病(例如疟疾、丝虫病、登革热等),其行为与人类生活息息相关.由于长久以来大量、广泛地使用杀虫剂,使蚊虫抗性日益严重.蚊虫的抗性机制主要有靶标抗性(包括神经轴突钠离子通道、乙酰胆碱酯酶和γ-氨基丁酸受体氯离子通道的突变)和代谢抗性(包括非特异性羧酸酯酶、细胞色素P450和谷胱甘肽-S-转移酶的活性增加)两方面.现对这些机制的研究进展进行综述,试图全面了解大量、广泛使用杀虫剂之后蚊虫抗性产生的分子机制.%Mosquitoes, which act as important medical insects, can transmit diseases such as malaria, fiariasis and dengue fever, etc. by bites, being harmful to humans. For a long period of time, due to the extensive use of chemical insecticides, the insecticide resistance in mosquitoes is becoming more and more serious. The mechanisms of resistance can be classified into two groups, knockdown resistance (such as saltations in sodium channel, Acetylcholinesterase and 7-aminobutyric acid) and metabolic resistance (such as augmentations in carboxylesterase, cytochrome P450 and glutathione-S-transferase). In this article, recent research advances in the resistance of mosquitoes to chemical insecticides are reviewed for better understanding of the molecular mechanism of mosquito resistance to the extensively used chemical insecticides.

  16. Biochemical Mechanism of Chlorantraniliprole Resistance in the Diamondback Moth, Plutella xylostella Linnaeus

    Institute of Scientific and Technical Information of China (English)

    HU Zhen-di; FENG Xia; LIN Qing-sheng; CHEN Huan-yu; LI Zhen-yu; YIN Fei; LIANG Pei; GAO Xi-wu

    2014-01-01

    The insecticide chlorantraniliprole exhibits good efifcacy and plays an important role in controlling the diamondback moth, Plutella xylostella Linnaeus. However, resistance to chlorantraniliprole has been observed recently in some ifeld populations. At present study, diamondback moths with resistance to chlorantraniliprole (resistant ratio (RR) was 82.18) for biochemical assays were selected. The assays were performed to determine potential resistance mechanisms. The results showed that the selected resistant moths (GDLZ-R) and susceptible moth could be synergized by known metabolic inhibitors such as piperonyl butoxide (PBO), triphenyl phosphate (TPP) and diethyl-maleate (DEM) at different levels (1.68-5.50-fold and 2.20-2.89-fold, respectively), and DEM showed the maximum synergism in both strains. In enzymes assays, a high level of glutathione-S-transferase (GST) was observed in the resistant moth, in contrast, moths that are susceptible to the insecticide had only 1/3 the GST activity of the resistant moths. The analysis of short-term exposure of chlorantraniliprole on biochemical response in the resistant strain also showed that GST activity was signiifcantly elevated after exposure to a sub-lethal concentration of chlorantraniliprole (about 1/3 LC50, 12 mg L-1) 12 and 24 h, respectively. The results show that there is a strong correlation between the enzyme activity and resistance, and GST is likely the main detoxiifcation mechanism responsible for resistance to chlorantraniliprole in P. xylostella L., cytochrome P450 monooxygenase (P450) and carboxy-lesterase (CarE) are involved in to some extent.

  17. Gene Polymorphisms and Chemotherapy in Non-small Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    Kayo OSAWA

    2009-01-01

    The phamacogenetics is being used to predict whether the selected chemotherapy will be really effective and tolerable to the patient. Irinotecan, oxidized by CYP3A4 to produce inactive compounds, is used for treatment of various cancers including advanced non small cell lung cancer (NSCLC) patients. CYP3A4*16B polymorphism was associated with decreased metabolism ofirrinotecan. Irinotecan is also metabolized by carboxylesterase to its principal active metabolite, SN-38, which is subsequently glucuronidated by UGT1As to form the inactive compound SN-38G. UGT1A1*28 and UGT1A1*6 polymorphisms were useful for predicting severe toxicity with NSCLC patients treated with irinotecan-based chemotherapy. Platinum-based compounds (cisplatin, carboplatin) are being used in combination with new cytotoxic drugs such as gemcitabine, paclitaxel, docetaxel, or vinorelbine in the treatment of advanced NSCLC. Cisplatin activity is mediated through the formation of cisplatin-DNA adducts. Gene polymorphisms of DNA repair factors are therefore obvious candidates for determinants of repair capacity and chemotherapy efficacy. ERCC1, XRCC1 and XRCC3 gene polymorphisms were a useful marker for predicting better survival in advanced NSCLC patients treated with platinum-based chemotherapy. XPA and XPD polymorphisms significantly increased response to platinum-based chemotherapy. These DNA repair gene polymorphisms were useful as a predictor of clinical outcome to the platinum-based chemotherapy. EGFR kinase inhibitors induce dramatic clinical responses in NSCLC patients with advanced disease. EGFR gene polymorphism in intron 1 contains a polymorphic single sequence dinudeotide repeat (CA-SSR) showed a statistically significant correlation with the gefitinib response and was appeared to be a useful predictive marker of the development of clinical outcome containing skin rashes with gefitinib treatment. The other polymorphisms of EGFR were also associated with increased EGFR promoter activity

  18. Gene Polymorphisms and Chemotherapy in Non-small Cell Lung Cancer

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    Kayo OSAWA

    2009-08-01

    Full Text Available The phamacogenetics is being used to predict whether the selected chemotherapy will be really effective and tolerable to the patient. Irinotecan, oxidized by CYP3A4 to produce inactive compounds, is used for treatment of various cancers including advanced non small cell lung cancer (NSCLC patients. CYP3A4*16B polymorphism was associated with decreased metabolism of irrinotecan. Irinotecan is also metabolized by carboxylesterase to its principal active metabolite, SN-38, which is subsequently glucuronidated by UGT1As to form the inactive compound SN-38G. UGT1A1*28 and UGT1A1*6 polymorphisms were useful for predicting severe toxicity with NSCLC patients treated with irinotecan-based chemotherapy. Platinum-based compounds (cisplatin, carboplatin are being used in combination with new cytotoxic drugs such as gemcitabine, paclitaxel, docetaxel, or vinorelbine in the treatment of advanced NSCLC. Cisplatin activity is mediated through the formation of cisplatin-DNA adducts. Gene polymorphisms of DNA repair factors are therefore obvious candidates for determinants of repair capacity and chemotherapy efficacy. ERCC1, XRCC1 and XRCC3 gene polymorphisms were a useful marker for predicting better survival in advanced NSCLC patients treated with platinum-based chemotherapy. XPA and XPD polymorphisms significantly increased response to platinum-based chemotherapy. These DNA repair gene polymorphisms were useful as a predictor of clinical outcome to the platinum-based chemotherapy. EGFR kinase inhibitors induce dramatic clinical responses in NSCLC patients with advanced disease. EGFR gene polymorphism in intron 1 contains a polymorphic single sequence dinucleotide repeat (CA-SSR showed a statistically significant correlation with the gefitinib response and was appeared to be a useful predictive marker of the development of clinical outcome containing skin rashes with gefitinib treatment. The other polymorphisms of EGFR were also associated with increased EGFR

  19. Gene expression in rats with Barrett's esophagus and esophageal adenocarcinoma induced by gastroduodenoesophageal reflux

    Institute of Scientific and Technical Information of China (English)

    Peng Cheng; Jun Gong; Tao Wang; Jie Chen; Gui-Sheng Liu; Ru Zhang

    2005-01-01

    AIM: To study the different gene expression profiles in rats with Barrett's esophagus (BE) and esophageal adenocarcinoma (EA) induced by gastro-duodenoesophageal reflux.METHODS: Esophagoduodenostomy was performed in 8-wk old Sprague-Dawley rats to induce gastro-duodenoesophageal reflux, and a group of rats that received sham operation served as control. Esophageal epithelial pathological tissues were dissected and frozen in liquid nitrogen immediately. The expression profiles of 4 096genes in EA and BE tissues were compared to normal esophagus epithelium in normal control (NC) by cDNA microarray.RESULTS: Four hundred and forty-eight genes in BE were more than three times different from those in NC, including 312 upregulated and 136 downregulated genes. Three hundred and seventy-seven genes in EA were more than three times different from those in NC, including 255upregulated and 142 downregulated genes. Compared to BE, there were 122 upregulated and 156 downregulated genes in EA. In the present study, the interested genes were those involved in carcinogenesis. Among them, the upregulated genes included cathepsin C, aminopeptidase M, arachidonic acid epoxygenase, tryptophan-2,3-dioxygenase, ubiquitin-conjugating enzyme, cyclic GMP-stimulated phosphodiesterase, tissue inhibitor of metalloproteinase-1, betaine-homocysteine methyltransferase, lysozyme, complement 4b binding protein,complement 9 protein, insulin-like growth factor binding protein, UDP-glucuronosyltransferase, tissue inhibitor of metalloproteinase-3, aldolase B, retinoid X receptor gamma, carboxylesterase and testicular cell adhesion molecule 1. The downregulated genes included glutathione synthetase, lecithin-cholesterol acyltransferase, p55CDC,heart fatty acid binding protein, cell adhesion regulator and endothelial cell selectin ligand.CONCLUSION: Esophageal epithelium exposed excessively to harmful ingredients of duodenal and gastric reflux may develop into BE and even EA gradually. The gene

  20. Permethrin-induced oxidative stress and toxicity and metabolism. A review.

    Science.gov (United States)

    Wang, Xu; Martínez, María-Aránzazu; Dai, Menghong; Chen, Dongmei; Ares, Irma; Romero, Alejandro; Castellano, Victor; Martínez, Marta; Rodríguez, José Luis; Martínez-Larrañaga, María-Rosa; Anadón, Arturo; Yuan, Zonghui

    2016-08-01

    Permethrin (PER), the most frequently used synthetic Type I pyrethroid insecticide, is widely used in the world because of its high activity as an insecticide and its low mammalian toxicity. It was originally believed that PER exhibited low toxicity on untargeted animals. However, as its use became more extensive worldwide, increasing evidence suggested that PER might have a variety of toxic effects on animals and humans alike, such as neurotoxicity, immunotoxicity, cardiotoxicity, hepatotoxicity, reproductive, genotoxic, and haematotoxic effects, digestive system toxicity, and cytotoxicity. A growing number of studies indicate that oxidative stress played critical roles in the various toxicities associated with PER. To date, almost no review has addressed the toxicity of PER correlated with oxidative stress. The focus of this article is primarily to summarise advances in the research associated with oxidative stress as a potential mechanism for PER-induced toxicity as well as its metabolism. This review summarises the research conducted over the past decade into the reactive oxygen species (ROS) generation and oxidative stress as a consequence of PER treatments, and ultimately their correlation with the toxicity and the metabolism of PER. The metabolism of PER involves various CYP450 enzymes, alcohol or aldehyde dehydrogenases for oxidation and the carboxylesterases for hydrolysis, through which oxidative stress might occur, and such metabolic factors are also reviewed. The protection of a variety of antioxidants against PER-induced toxicity is also discussed, in order to further understand the role of oxidative stress in PER-induced toxicity. This review will throw new light on the critical roles of oxidative stress in PER-induced toxicity, as well as on the blind spots that still exist in the understanding of PER metabolism, the cellular effects in terms of apoptosis and cell signaling pathways, and finally strategies to help to protect against its oxidative

  1. Enantiospecific determination of DL-methylphenidate and DL-ethylphenidate in plasma by liquid chromatography-tandem mass spectrometry: application to human ethanol interactions.

    Science.gov (United States)

    Zhu, Hao-Jie; Patrick, Kennerly S; Markowitz, John S

    2011-04-01

    In humans, concomitant DL-methylphenidate (DL-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite DL-ethylphenidate (DL-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between D- and L-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties. Accordingly, a sensitive, accurate and precise enantiospecific analytical method is required in order to fully explore pharmacokinetic-pharmacodynamic correlations regarding the MPH-ethanol interaction. The present study describes a novel liquid chromatographic-tandem mass spectrometric method for simultaneous analysis of D- and L-MPH as well as D- and L-EPH concentrations from human plasma. This assay provides baseline resolution of the individual MPH and EPH isomers utilizing a vancomycin-based chiral column. The lower limit of quantification was 0.025 ng/ml for each isomer when extracting 0.5 ml plasma aliquots. Calibration curves were linear over the range from 0.025 ng/ml to 25 ng/ml for all analytes (r(2)>0.995). Assay accuracy and precision were excellent and stability studies and assessment of potential matrix effects contributed to the validation of the method. Application of the method to human plasma samples collected after the administration of dl-MPH with or without ethanol is included, and the implications of this pharmacokinetic drug interaction discussed.

  2. Carboxylic Esterase and Its Associations With Long-term Effects of Organophosphorus Pesticides

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To examine a) the effect of organophosphorus pesticide exposure on activity of carboxylic esterases, namely butyrylcholinesterase (BChE), carboxylesterase (CarbE) and paraoxonase (PonE); and b) the association of polymorphisms of BChE and PonE with individual genetic susceptibility to organophosphorus pesticide exposure. Methods A cross-sectional study was conducted in 75 workers exposed to organophosphorus pesticides and 100 non-exposed controls. The serum activity of these enzymes was measured. Variant forms of BCHE-K, PON-192, and PON-55 were detected. A symptom score was developed as a proxy measure of clinical outcomes. Results Activities of both BChE and CarbE were lower in exposed exposed workers with BCHE-K genotype UU (61 cases), genotype UK (12 cases) and genotype KK (2 cases) was 105.05, 84.42 activity in the exposed workers with PON-192 genotype BB (37), genotype AB (27) and genotype AA (11) was 116.8, 91.2, and 9.20. The symptom score was the highest in individuals with abnormal homozygote for each of the three gene loci. Conclusions Long-term exposure to organophosphorus pesticides can inhibit BChE and CarbE activity, but exerts no inhibitory effect on PonE activity. Different genotypes of BCHE-K, PON-192, and PON-55 may be related to the severity of adverse health effects of organophosphorus pesticide exposure. Implications of potentially higher susceptibility of workers with mutant homozygotes should be evaluated to reduce health risks.

  3. Comparative assessment of in vitro and in vivo toxicity of azinphos methyl and its commercial formulation.

    Science.gov (United States)

    Güngördü, Abbas; Uçkun, Miraç

    2015-09-01

    The toxic effects of Gusathion (GUS), which is a commercial organophosphate (OP) pesticide, and also its active ingredient, azinphos methyl (AzM), are evaluated comparatively with in vitro and in vivo studies. Initially, the 96-h LC50 values of AzM and GUS were estimated for two different life stages of Xenopus laevis, embryos, and tadpoles. The actual AzM concentrations in exposure media were monitored by high-performance liquid chromatography. Also, the sub-lethal effects of these compounds to tadpoles were determined 24 h later at exposure concentrations of 0.1 and 1 mg/L using selected biomarker enzymes such as acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione S-transferase (GST), glutathione reductase, lactate dehydrogenase, and aspartate aminotrasferase. Differences in AChE inhibition capacities of AzM and GUS were evaluated under in vitro conditions between frogs and fish in the second part of this study. The AChE activities in a pure electrical eel AChE solution and in brain homogenates of adult Cyprinus carpio, Pelophylax ridibundus, and X. laevis were assayed after in vitro exposure to 0.05, 0.5, 5, and 50 mg/L concentrations of AzM and GUS. According to in vivo studies AChE, CaE and GST are important biomarkers of the effect of OP exposure while CaE may be more effective in short-term, low-concentration exposures. The results of in vitro studies showed that amphibian brain AChEs were relatively more resistant to OP exposure than fish AChEs. The resistance may be the cause of the lower toxicity/lethality of OP compounds to amphibians than to fish.

  4. Insecticide resistance in the dengue vector Aedes aegypti from Martinique: distribution, mechanisms and relations with environmental factors.

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    Sébastien Marcombe

    Full Text Available Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies. The viruses responsible for dengue are transmitted by Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the "knock-down resistance" V1016I Kdr mutation at high frequency (>87%. Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed.

  5. Temephos resistance in Aedes aegypti in Colombia compromises dengue vector control.

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    Nelson Grisales

    Full Text Available BACKGROUND: Control and prevention of dengue relies heavily on the application of insecticides to control dengue vector mosquitoes. In Colombia, application of the larvicide temephos to the aquatic breeding sites of Aedes aegypti is a key part of the dengue control strategy. Resistance to temephos was recently detected in the dengue-endemic city of Cucuta, leading to questions about its efficacy as a control tool. Here, we characterize the underlying mechanisms and estimate the operational impact of this resistance. METHODOLOGY/PRINCIPAL FINDINGS: Larval bioassays of Ae. aegypti larvae from Cucuta determined the temephos LC50 to be 0.066 ppm (95% CI 0.06-0.074, approximately 15× higher than the value obtained from a susceptible laboratory colony. The efficacy of the field dose of temephos at killing this resistant Cucuta population was greatly reduced, with mortality rates <80% two weeks after application and <50% after 4 weeks. Neither biochemical assays nor partial sequencing of the ace-1 gene implicated target site resistance as the primary resistance mechanism. Synergism assays and microarray analysis suggested that metabolic mechanisms were most likely responsible for the temephos resistance. Interestingly, although the greatest synergism was observed with the carboxylesterase inhibitor, DEF, the primary candidate genes from the microarray analysis, and confirmed by quantitative PCR, were cytochrome P450 oxidases, notably CYP6N12, CYP6F3 and CYP6M11. CONCLUSIONS/SIGNIFICANCE: In Colombia, resistance to temephos in Ae. aegypti compromises the duration of its effect as a vector control tool. Several candidate genes potentially responsible for metabolic resistance to temephos were identified. Given the limited number of insecticides that are approved for vector control, future chemical-based control strategies should take into account the mechanisms underlying the resistance to discern which insecticides would likely lead to the greatest

  6. A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao

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    Bailey Bryan A

    2008-11-01

    Full Text Available Abstract Background The basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' Broom Disease (WBD in cacao (Theobroma cacao. It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. M. perniciosa, together with the related species M. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9× coverage of M. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen. Results Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models indicates that M. perniciosa has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin. Analysis of gene families indicated that M. perniciosa have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in M. perniciosa genome survey. Conclusion This genome survey gives an overview of the M. perniciosa genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the M. perniciosa

  7. Involvement of Three Esterase Genes from Panonychus citri (McGregor in Fenpropathrin Resistance

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    Xiao-Min Shen

    2016-08-01

    Full Text Available The citrus red mite, Panonychus citri (McGregor, is a major citrus pest with a worldwide distribution and an extensive record of pesticide resistance. However, the underlying molecular mechanism associated with fenpropathrin resistance in this species have not yet been reported. In this study, synergist triphenyl phosphate (TPP dramatically increased the toxicity of fenpropathrin, suggesting involvement of carboxylesterases (CarEs in the metabolic detoxification of this insecticide. The subsequent spatiotemporal expression pattern analysis of PcE1, PcE7 and PcE9 showed that three CarEs genes were all over-expressed after insecticide exposure and higher transcripts levels were observed in different field resistant strains of P. citri. Heterologous expression combined with 3-(4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetra-zolium bromide (MTT cytotoxicity assay in Spodoptera frugiperda (Sf9 cells revealed that PcE1-, PcE7- or PcE9-expressing cells showed significantly higher cytoprotective capability than parental Sf9 cells against fenpropathrin, demonstrating that PcEs probably detoxify fenpropathrin. Moreover, gene silencing through the method of leaf-mediated dsRNA feeding followed by insecticide bioassay increased the mortalities of fenpropathrin-treated mites by 31% (PcE1, 27% (PcE7 and 22% (PcE9, respectively, after individual PcE gene dsRNA treatment. In conclusion, this study provides evidence that PcE1, PcE7 and PcE9 are functional genes mediated in fenpropathrin resistance in P. citri and enrich molecular understanding of CarEs during the resistance development of the mite.

  8. Standard Versus Continuous Administration of Capecitabine in Metastatic Breast Cancer (GEICAM/2009-05): A Randomized, Noninferiority Phase II Trial With a Pharmacogenetic Analysis

    Science.gov (United States)

    Martínez, Noelia; Ramos, Manuel; Calvo, Lourdes; Lluch, Ana; Zamora, Pilar; Muñoz, Montserrat; Carrasco, Eva; Caballero, Rosalía; García-Sáenz, José Ángel; Guerra, Eva; Caronia, Daniela; Casado, Antonio; Ruíz-Borrego, Manuel; Hernando, Blanca; Chacón, José Ignacio; De la Torre-Montero, Julio César; Jimeno, María Ángeles; Heras, Lucía; Alonso, Rosario; De la Haba, Juan; Pita, Guillermo; Constenla, Manuel; González-Neira, Anna

    2015-01-01

    Background. The approved capecitabine regimen as monotherapy in metastatic breast cancer (MBC) is 1,250 mg/m2 twice daily for 2 weeks on and 1 week off (Cint). Dose modifications are often required because of severe hand-foot syndrome (HFS). We tested a continuous regimen with a lower daily dose but a similar cumulative dose in an attempt to reduce the severity of adverse events (AEs) while maintaining efficacy. Methods. We randomized 195 patients with HER-2/neu-negative MBC to capecitabine 800 mg/m2 twice daily throughout the 21-day cycle (Ccont) or to Cint to assess noninferiority in the percentage of patients free of progression at 1 year. Secondary endpoints included efficacy and safety. Associations between polymorphisms in capecitabine metabolism-related genes and drug response were assessed. Results. The percentage of patients free of progression at 1 year was 27.3% with Cint versus 25.3% with Ccont (difference of −2.0%; 95% confidence interval: −15.5% to 11.5%, exceeding the 15% deemed noninferior). Differences regarding other efficacy variables were also not found. Grade 3–4 HFS was the most frequent AE (41.1% in Cint vs. 42.3% in Ccont). Grade 3–4 neutropenia, thrombocytopenia, diarrhea, and stomatitis were more frequent with Cint. A 5′ untranslated region polymorphism in the carboxylesterase 2 gene was associated with HFS. One polymorphism in cytidine deaminase and two in thymidine phosphorylase were associated with survival. Conclusion. Our study was unable to show noninferiority with the continuous capecitabine regimen (Ccont) compared with the approved intermittent regimen (Cint). Further investigation is required to improve HFS. Polymorphisms in several genes might contribute to interindividual differences in response to capecitabine. PMID:25601966

  9. Vinyl acetate monomer (VAM) genotoxicity profile: relevance for carcinogenicity.

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    Albertini, Richard J

    2013-09-01

    Vinyl acetate monomer (VAM) is a site-of-contact carcinogen in rodents. It is also DNA reactive and mutagenic, but only after its carboxylesterase mediated conversion to acetaldehyde (AA), a metabolic reaction that also produces acetic acid and protons. As VAM's mutagenic metabolite, AA is normally produced endogenously; detoxification by aldehyde dehydrogenase (ALDH) is required to maintain intra-cellular AA homeostasis. This review examines VAM's overall genotoxicity, which is due to and limited by AA, and the processes leading to mutation induction. VAM and AA have both been universally negative in mutation studies in bacteria but both have tested positive in several in vitro studies in higher organisms that usually employed high concentrations of test agents. Recently however, in vitro studies evaluating submillimolar concentrations of VAM or AA have shown threshold dose-responses for mutagenicity in human cultured cells. Neither VAM nor AA induced systemic mutagenicity in in vivo studies in metabolically competent mice when tested at non-lethal doses while treatments of animals deficient in aldehyde dehydrogenase (Aldh in animals) did induce both gene and chromosome level mutations. The results of several studies have reinforced the critical role for aldehyde dehydrogenase 2 (ALDH2 in humans) in limiting AA's (and therefore VAM's) mutagenicity. The overall aim of this review of VAM's mutagenic potential through its AA metabolite is to propose a mode of action (MOA) for VAM's site-of-contact carcinogenesis that incorporates the overall process of mutation induction that includes both background mutations due to endogenous AA and those resulting from exogenous exposures.

  10. Nonlinear responses for chromosome and gene level effects induced by vinyl acetate monomer and its metabolite, acetaldehyde in TK6 cells.

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    Budinsky, Robert; Gollapudi, Bhaskar; Albertini, Richard J; Valentine, Rudolph; Stavanja, Mari; Teeguarden, Justin; Fensterheim, Robert; Rick, David; Lardie, Thomas; McFadden, Lisa; Green, Amanda; Recio, Leslie

    2013-12-01

    Vinyl acetate monomer (VAM) produced rat nasal tumors at concentrations in the hundreds of parts per million. However, VAM is weakly genotoxic in vitro and shows no genotoxicity in vivo. A European Union Risk Assessment concluded that VAM's hydrolysis to acetaldehyde (AA), via carboxylesterase, is a critical key event in VAM's carcinogenic potential. In the following study, we observed increases in micronuclei (MN) and thymidine kinase (Tk) mutants that were dependent on the ability of TK6 cell culture conditions to rapidly hydrolyze VAM to AA. Heat-inactivated horse serum demonstrated a high capacity to hydrolyze VAM to AA; this activity was highly correlated with a concomitant increase in MN. In contrast, heat-inactivated fetal bovine serum (FBS) did not hydrolyze VAM and no increase in MN was observed. AA's ability to induce MN was not impacted by either serum since it directly forms Schiff bases with DNA and proteins. Increased mutant frequency at the Tk locus was similarly mitigated when AA formation was not sufficiently rapid, such as incubating VAM in the presence of FBS for 4 hr. Interestingly, neither VAM nor AA induced mutations at the HPRT locus. Finally, cytotoxicity paralleled genotoxicity demonstrating that a small degree of cytotoxicity occurred prior to increases in MN. These results established 0.25 mM as a consistent concentration where genotoxicity first occurred for both VAM and AA provided VAM is hydrolyzed to AA. This information further informs significant key events related to the mode of action of VAM-induced nasal mucosal tumors in rats.

  11. Effect of High Fructose Syrup Diet Exposure on the Activities of Detoxifying Enzymes in Honey Bees Apis mellifera ligustica%饲喂果葡糖浆对意大利蜜蜂解毒酶的影响

    Institute of Scientific and Technical Information of China (English)

    孟丽峰; 靳三省; 刁青云

    2013-01-01

    为了探讨果葡糖浆饲喂蜜蜂的安全性,以意大利蜜蜂(Apis mellifera ligustica)为实验材料,蔗糖作为对照,饲喂果葡糖浆2个月后,检测意大利蜜蜂体内解毒酶的变化情况.结果表明:饲喂果葡糖浆后,意大利蜜蜂大幼虫体内细胞色素P450比活力、成年工蜂腹部谷胱甘肽-S-转移酶和羧酸酯酶比活力均与对照无显著差异.短期饲喂果葡糖浆对蜜蜂是安全的,长期影响还有待于继续研究.%The activities of Cytochrome-P450,glutathione S-transferase and carboxylesterase in the worker bees of Apis mellifera ligustica were investigated after the bees were fed orally with high fructose syrup in two consecutive months.The results showed that compared with sucrose diet,high fructose syrup diet did not significantly affect the activities of three detoxifying enzymes.Feeding with high fructose syrups is safe to Apis mellifera ligustica in short time and long-time effects need further research.The results can be used to assess the security of high fructose syrups used as bee feed.

  12. Effects of dexamethasone coadministered with oseltamivir on the pharmacokinetics of oseltamivir in healthy volunteers

    Science.gov (United States)

    Jang, Kyungho; Kim, Min-Kyoung; Oh, Jaeseong; Lee, SeungHwan; Cho, Joo-Youn; Yu, Kyung-Sang; Choi, Tai Kiu; Lee, Sang-Hyuk; Lim, Kyoung Soo

    2017-01-01

    Purpose Oseltamivir is widely used in the treatment and prophylaxis of influenza A and B viral infections. It is ingested as an oral prodrug that is rapidly metabolized by carboxylesterase 1 (CES1) to its active form, oseltamivir carboxylate. Dexamethasone is also used in the treatment of acute respiratory distress syndrome, a severe complication of influenza; however, its influence on the pharmacokinetics (PK) of oseltamivir is controversial. The aim of this study was to investigate the effects of coadministering oseltamivir and dexamethasone on the PK of oseltamivir in healthy volunteers. Methods An open-label, two-period, one-sequence, multiple-dose study was conducted in 19 healthy male volunteers. Oseltamivir (75 mg) was orally administered on Day 1 and Day 8, and dexamethasone (1.5 mg) was administered once daily from Day 3 to Day 8. Serial blood and urine samples were collected for PK analysis of oseltamivir and oseltamivir carboxylate on Day 1 and Day 8. Oseltamivir and oseltamivir carboxylate concentrations in plasma and urine were determined using liquid chromatography–tandem mass spectrometry. Results Area under the plasma concentration–time curve (AUC) of oseltamivir and oseltamivir carboxylate decreased after dexamethasone treatment for 6 days. The geometric mean ratio (90% confidence interval) of the metabolic ratio (oseltamivir carboxylate AUC0–48h/oseltamivir AUC0–48h) was 0.92 (0.87–0.97). The amount of unchanged oseltamivir excreted in urine increased by 14% after dexamethasone treatments. Conclusion Coadministration of dexamethasone with oseltamivir slightly decreased systemic exposure to oseltamivir and oseltamivir carboxylate in healthy volunteers. This result suggests that CES1 is inhibited by dexamethasone in humans. However, coadministration of oseltamivir and dexamethasone did not appear to have a clinically relevant effect on the PK of oseltamivir; based on these results, dexamethasone can be coadministered with oseltamivir. PMID

  13. Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: supersensitivity of acetylcholinesterase knockout mouse to VX lethality.

    Science.gov (United States)

    Duysen, E G; Li, B; Xie, W; Schopfer, L M; Anderson, R S; Broomfield, C A; Lockridge, O

    2001-11-01

    The possibility that organophosphate toxicity is due to inhibition of targets other than acetylcholinesterase (AChE, EC 3.1.1.7) was examined in AChE knockout mice. Mice (34-55 days old) were grouped for this study, after it was determined that AChE, butyrylcholinesterase (BChE), and carboxylesterase activities had reached stable values by this age. Mice with 0, 50, or 100% AChE activity were treated subcutaneously with the nerve agent VX. The LD50 for VX was 10 to 12 microg/kg in AChE-/-, 17 microg/kg in AChE+/-, and 24 microg/kg in AChE+/+ mice. The same cholinergic signs of toxicity were present in AChE-/- mice as in wild-type mice, even though AChE-/- mice have no AChE whose inhibition could lead to cholinergic signs. Wild-type mice, but not AChE-/- mice, were protected by pretreatment with atropine. Tissues were extracted from VX-treated and untreated animals and tested for AChE, BChE, and acylpeptide hydrolase activity. VX treatment inhibited 50% of the AChE activity in brain and muscle of AChE+/+ and +/- mice, 50% of the BChE activity in all three AChE genotypes, but did not significantly inhibit acylpeptide hydrolase activity. It was concluded that the toxicity of VX must be attributed to inhibition of nonacetylcholinesterase targets in the AChE-/- mouse. Organophosphorus ester toxicity in wild-type mice is probably due to inhibition or binding to several proteins, only one of which is AChE.

  14. An unexpected plasma cholinesterase activity rebound after challenge with a high dose of the nerve agent VX.

    Science.gov (United States)

    Dorandeu, F; Foquin, A; Briot, R; Delacour, C; Denis, J; Alonso, A; Froment, M T; Renault, F; Lallement, G; Masson, P

    2008-06-27

    Organophosphorus chemical warfare agents (nerve agents) are to be feared in military operations as well as in terrorist attacks. Among them, VX (O-ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothioate) is a low volatility liquid that represents a percutaneous as well as an inhalation hazard if aerosolized. It is a potent irreversible cholinesterase (ChE) inhibitor that causes severe signs and symptoms, including respiratory dysfunction that stems from different mechanisms. VX-induced pulmonary oedema was previously reported in dogs but mechanisms involved are not well understood, and its clinical significance remains to be assessed. An experimental model was thus developed to study VX-induced cardiovascular changes and pulmonary oedema in isoflurane-anaesthetized swine. In the course of this study, we observed a fast and unexpected rebound of plasma ChE activity following inhibition provoked by the intravenous injection of 6 and 12 microg kg(-1) of VX. In whole blood ChE activity, the rebound could stay unnoticed. Further investigations showed that the rebound of plasma esterase activity was neither related to spontaneous reactivation of ChE nor to VX-induced increase in paraoxonase/carboxylesterase activities. A bias in Ellman assay, haemoconcentration or severe liver cytolysis were also ruled out. All in all, these results suggest that the rebound was likely due to the release of butyrylcholinesterase into the blood stream from ChE producing organs. Nature of the organ(s) and mechanisms involved in enzyme release will need further investigations as it may represent a mechanism of defence, i.e. VX scavenging, that could advantageously be exploited.

  15. Insecticide resistance to organophosphates in Culex pipiens complex from Lebanon

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    Osta Mike A

    2012-07-01

    Full Text Available Abstract Background Analysis of Culex pipiens mosquitoes collected from a single site in Lebanon in 2005, revealed an alarming frequency of ace-1 alleles conferring resistance to organophosphate insecticides. Following this, in 2006 the majority of municipalities switched to pyrethroids after a long history of organophosphate usage in the country; however, since then no studies have assessed the impact of changing insecticide class on the frequency of resistant ace-1 alleles in C. pipiens. Methods C. pipiens mosquitoes were captured indoors from 25 villages across the country and subjected to established methods for the analysis of gene amplification at the Ester locus and target site mutations in ace-1 gene that confer resistance to organophosphates. Results We conducted the first large-scale screen for resistance to organosphosphates in C. pipiens mosquitoes collected from Lebanon. The frequency of carboxylesterase (Ester and ace-1 alleles conferring resistance to organophosphates were assessed among C. pipiens mosquitoes collected from 25 different villages across the country between December 2008 and December 2009. Established enzymatic assay and PCR-based molecular tests, both diagnostic of the major target site mutations in ace-1 revealed the absence of the F290V mutation among sampled mosquitoes and significant reduction in the frequency of G119S mutation compared to that previously reported for mosquitoes collected from Beirut in 2005. We also identified a new duplicated ace-1 allele, named ace-1D13, exhibiting a resistant phenotype by associating a susceptible and a resistant copy of ace-1 in a mosquito line sampled from Beirut in 2005. Fisher’s exact test on ace-1 frequencies in the new sample sites, showed that some populations exhibited a significant excess of heterozygotes, suggesting that the duplicated allele is still present. Starch gel electrophoresis indicated that resistance at the Ester locus was mainly attributed to the

  16. Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies

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    Yébakima André

    2009-10-01

    Full Text Available Abstract Background The yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood. Results The present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies. Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71% of the sodium channel 'knock down resistance' (kdr mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT

  17. Characterization, sub-cellular localization and expression profiling of the isoprenylcysteine methylesterase gene family in Arabidopsis thaliana

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    Ma Wujun

    2010-09-01

    Full Text Available Abstract Background Isoprenylcysteine methylesterases (ICME demethylate prenylated protein in eukaryotic cell. Until now, knowledge about their molecular information, localization and expression pattern is largely unavailable in plant species. One ICME in Arabidopsis, encoded by At5g15860, has been identified recently. Over-expression of At5g15860 caused an ABA hypersensitive phenotype in transgenic Arabidopsis plants, indicating that it functions as a positive regulator of ABA signaling. Moreover, ABA induced the expression of this gene in Arabidopsis seedlings. The current study extends these findings by examining the sub-cellular localization, expression profiling, and physiological functions of ICME and two other ICME-like proteins, ICME-LIKE1 and ICME-LIKE2, which were encoded by two related genes At1g26120 and At3g02410, respectively. Results Bioinformatics investigations showed that the ICME and other two ICME-like homologs comprise a small subfamily of carboxylesterase (EC 3.1.1.1 in Arabidopsis. Sub-cellular localization of GFP tagged ICME and its homologs showed that the ICME and ICME-like proteins are intramembrane proteins predominantly localizing in the endoplasmic reticulum (ER and Golgi apparatus. Semi-quantitative and real-time quantitative PCR revealed that the ICME and ICME-like genes are expressed in all examined tissues, including roots, rosette leaves, cauline leaves, stems, flowers, and siliques, with differential expression levels. Within the gene family, the base transcript abundance of ICME-LIKE2 gene is very low with higher expression in reproductive organs (flowers and siliques. Time-course analysis uncovered that both ICME and ICME-like genes are up-regulated by mannitol, NaCl and ABA treatment, with ICME showing the highest level of up-regulation by these treatments. Heat stress resulted in up-regulation of the ICME gene significantly but down-regulation of the ICME-LIKE1 and ICME-LIKE2 genes. Cold and dehydration

  18. RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

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    Mamidala Praveen

    2012-01-01

    Full Text Available Abstract Background Bed bugs (Cimex lectularius are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. Results We performed a next-generation RNA sequencing (RNA-Seq experiment to find differentially expressed genes between pesticide-resistant (PR and pesticide-susceptible (PS strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs and our previous 454 pyrosequenced database (21,088 ESTs. The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2 revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. Conclusions We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide

  19. Esterases of Varroa destructor (Acari: Varroidae), parasitic mite of the honeybee.

    Science.gov (United States)

    Dmitryjuk, Małgorzata; Żołtowska, Krystyna; Frączek, Regina; Lipiński, Zbigniew

    2014-04-01

    Varroa destructor is an ectoparasite that causes serious damage to the population of the honeybee. Increasing resistance of the parasite to acaricides is related, among others, to metabolic adaptations of its esterases to facilitate decomposition of the chemicals used. Esterases are a large heterogeneous group of enzymes that metabolize a number of endogenous and exogenous substrates with ester binding. The aim of the present study was to determine the activity of esterases in the body extracts (BE) and excretion/secretion products (E/SP) of the mite. The enzymes contained in the E/SP should originate mainly from the salivary glands and the alimentary system and they may play a particularly important role in the first line of defence of the mite against acaricides. Activity of cholinesterases (ChEs) [acetylcholinesterase (AChE) and butyrylcholinesterase], carboxylesterases (CEs) and phosphatases [alkaline phosphatase (AP) and acid phosphatase (AcP)] was investigated. The activity of all the enzymes except AChE was higher in the E/SP than in the BE. ChEs from the BE and from the E/SP reacted differently on eserine, a ChE inhibitor. Eserine inhibited both enzymes from the BE, increased decomposition of acetylcholine, but did not influence hydrolysis of butyrylcholine by the E/SP. Activity of the CEs from the BE in relation to the esters of carboxylic acids can be presented in the following series: C10 > C12 > C14 > C8 > C2 > C4 = C16, while activity of the CEs from the E/SP was: C4 > C8 > C2 > C14 > C10 > C12 > C16. The inhibitor of CEs, triphenyl phosphate, reduced the activity of esterases C2–C8 and C14–C16; however, it acted in the opposite way to CEs C10 and C12. The activity of both phosphatases was higher in the E/SP than in the BE (AcP about twofold and AP about 2.6-fold); the activities of AP and AcP in the same material were similar. Given the role of esterases in resistance to pesticides, further studies are necessary to obtain complete biochemical

  20. Comparative Study of Malathion Toxicity and General Esterases in Larvae and Adults from a Field Population of Oxya chinensis (Thunberg)(Orthoptera:Acridoidea)

    Institute of Scientific and Technical Information of China (English)

    WU Hai-hua; YANG Mei-ling; GUO Ya-ping; MA En-bo

    2004-01-01

    both larvae and adults. From the analysis of inhibition in vitro, the esterases in the two life stages were B-type,and carboxylesterases were predominant enzymes in the composition of the esterases in the two stages.

  1. Insecticide resistance and, efficacy of space spraying and larviciding in the control of dengue vectors Aedes aegypti and Aedes albopictus in Sri Lanka.

    Science.gov (United States)

    Karunaratne, S H P P; Weeraratne, T C; Perera, M D B; Surendran, S N

    2013-09-01

    Unprecedented incidence of dengue has been recorded in Sri Lanka in recent times. Source reduction and use of insecticides in space spraying/fogging and larviciding, are the primary means of controlling the vector mosquitoes Aedes aegypti and Ae. albopictus in the island nation. A study was carried out to understand insecticide cross-resistance spectra and mechanisms of insecticide resistance of both these vectors from six administrative districts, i.e. Kandy, Kurunegala, Puttalam, Gampaha, Ratnapura and Jaffna, of Sri Lanka. Efficacy of the recommended dosages of frequently used insecticides in space spraying and larviciding in dengue vector control programmes was also tested. Insecticide bioassay results revealed that, in general, both mosquito species were highly resistant to DDT but susceptible to propoxur and malathion except Jaffna Ae. aegypti population. Moderate resistance to malathion shown by Jaffna Ae. aegypti population correlated with esterase and malathion carboxylesterase activities of the population. High levels of acetylcholinesterase (AChE) insensitivity in the absence of malathion and propoxur resistance may be due to non-synaptic forms of AChE proteins. Moderate pyrethroid resistance in the absence of high monooxygenase levels indicated the possible involvement of 'kdr' type resistance mechanism in Sri Lankan dengue vectors. Results of the space spraying experiments revealed that 100% mortality at a 10 m distance and >50% mortality at a 50 m distance can be achieved with malathion, pesguard and deltacide even in a ground with dense vegetation. Pesguard and deltacide spraying gave 100% mortality up to 50 m distance in open area and areas with little vegetation. Both species gave >50% mortalities for deltacide at a distance of 75 m in a dense vegetation area. Larval bioassays conducted in the laboratory showed that a 1 ppm temephos solution can maintain a larval mortality rate of 100% for ten months, and the mortality rate declined to 0% in the

  2. Invasive mechanism and manasement stratesy of Bemisia tabaci (Gennadius) biotype B: Progress report of 973 Program on invasive alien species in China

    Institute of Scientific and Technical Information of China (English)

    WAN FangHao; CUI XuHong; ZHANG LiPing; ZHANG Fan; ZHANG QingWen; LIU WanXue; LIANG Pei; LEI ZhongRen; ZHANG YongJun; ZHANG GuiFen; LIU ShuSheng; LUO Chen; CHU Dong; ZHANG YouJun; ZANG LianSheng; JIU Min; Lǖ ZhiChuang

    2009-01-01

    Bemisia tabaci (Gennadius) biotype B, called a "superbug", is one of the most harmful biotypes of this species complex worldwide. In this report, the invasive mechanism and management of B. tabaci bio-type B, based on our 5-year studies, are presented. Six B. tabaci biotypes, B, Q, ZHJ1, ZHJ2, ZHJ3 and FJ1, have been identified in China. Biotype B dominates the other biotypes in many regions of the country. Genetic diversity in biotype B might be induced by host plant, geographical conditions, and/or insecticidal application. The activities of CarE (carboxylesterase) and GSTs (glutathione-S-transferase) in biotype B reared on cucumber and squash were greater than on other host plants, which might have increased its resistance to insecticides. The higher activities of detoxification enzymes in biotype B might be induced by the secondary metabolites in host plants. Higher adaptive ability of biotype B adults to adverse conditions might be linked to the expression of heat shock protein genes. The in-digenous B. tabaci biotypes were displaced by the biotype B within 225 d. The asymmetric mating in-teractions and mutualism between biotype B and begomoviruses via its host plants speed up wide-spread invasion and displacement of other biotypes. B. tabaci biotype B displaced Trialeurodes vapo-rariorum (Westwood) after 4-7 generations under glasshouse conditions. Greater adaptive ability of the biotype B to adverse conditions and its rapid population increase might be the reasons of its suc-cessful displacement of T. vaporariorum. Greater ability of the biotype B to switch to different host plants may enrich its host plants, which might enable it to better compete with T. vaporariorum. Native predatory natural enemies possess greater ability to suppress B. tabaci under field conditions. The kairomones in the 3rd and 4th instars of biotype B may provide an important stimulus in host searching and location by its parasitoids. The present results provide useful information in

  3. Bio-synthesis and hydrolysis of ethyl phenylacetate and ethyl 2-phenylpropionate in organic solvent by lyophilized mycelia Biossíntese e hidrólise de fenilacetato de etila e 2-fenilpropionato de etila em solvente orgânico por meio de micélios liofilizados

    Directory of Open Access Journals (Sweden)

    Paolo Torre

    2007-06-01

    Full Text Available To select the best biocatalysts for ethanol acylations with phenylacetic and 2-phenylpropionic acids, lyophilized mycelia of Aspergillus oryzae CBS 10207, A. oryzae MIM, Rhizopus oryzae CBS 11207, R. oryzae CBS 39134, R. oryzae CBS 26028 and R. oryzae CBS 32847 were tested in this study. The carboxylesterase activities of A. oryzae MIM and R. oryzae 11207, which revealed to be the best biocatalysts, were investigated either in 0.1 M phosphate buffer or in n-heptane to catalyze the hydrolysis or the synthesis of ethyl esters of these acids, respectively. A. oryzae proved more effective than R. oryzae, probably due to more favorable microenvironment conditions and thermodynamic scenario. The results in terms of product formation and substrate consumption versus time were used to estimate the maximum conversion yields, the equilibrium constants and the times needed to reach half maximum conversion, thus providing sufficient information about these equilibria.Micélios liofilizados de Aspergillus oryzae CBS 10207, A. oryzae MIM, Rhizopus oryzae CBS 11207, R. oryzae CBS 39134, R. oryzae CBS 26028 e R. oryzae CBS 32847 foram testados neste estudo com vista à seleção do melhor biocatalisador para efetuar a acilação de etanol com ácidos fenilacético e 2-fenilpropiônico. As atividades carboxilesterásicas de A. oryzae MIM e R. oryzae 11207, que resultaram ser os melhores biocatalisadores, foram investigadas tanto em tampão fosfato 0,1 M como em n-heptano para catalisar a hidrólise ou a síntese dos ésteres etílicos destes ácidos. A. oryzae pareceu ser mais eficaz que R. oryzae, provavelmente devido a condições micro-ambientais e a um cenário termodinâmico mais favoráveis. Os resultados obtidos em termos de formação do produto e consumo dos substratos em função do tempo foram usados para a estimativa dos rendimentos de conversão máximos, as constantes de equilíbrio e os tempos necessários para alcançar metade da conversão m

  4. Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.

    Science.gov (United States)

    Carvalho, Renato A; Omoto, Celso; Field, Linda M; Williamson, Martin S; Bass, Chris

    2013-01-01

    The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide

  5. 人工饲料对草地螟消化酶活性及羧酸酯酶mRNA表达量的影响%Effects of Artificial Diets on Digestive Enzyme Activities in Loxostege sticticalis

    Institute of Scientific and Technical Information of China (English)

    吴晋华; 刘爱萍; 徐林波; 高书晶; 韩冰; 康爱国; 张玉慧

    2012-01-01

    通过对经验型筛选和正交试验优化出的饲料配方饲养的草地螟(Loxostege sticticalis)5龄幼虫中肠淀粉酶、蛋白酶、酯酶和脂肪酶4种消化酶进行活性测定.结果表明:饲料HG在蔗糖的添加量稍做改动后,其饲养效果更接近于草地螟天然食料灰菜(Chenopodium album);饲料12的淀粉酶、蛋白酶、酯酶和脂肪酶都与对照组相差较多,蛋白含量、脂类含量与糖类含量需做进一步的修正与试验.另外利用RT-PCR方法,通过研究人工饲料HG,12及对照组H对草地螟羧酸酯酶基因转录水平的影响,从分子水平快速评价出饲料HG优于饲料12.研究结果为今后的草地螟人工饲养及饲料配方进一步改进提供了理论依据.%This paper studied the bowel amylase, protease, ester enzyme and lipase of Loxostege sticticalis 5-instar larva fed by optimal feed formula which was selected by experience screening and orthogonal test. Results showed that the feed effect of artificial diet HG after adding sucrose was closer to that of Chenopo-dium album. The amylase, protease, esterase and lipase activities of artificial diet 12 were different from those of control group. In addition, the effects of artificial diet HG and 12 on carboxylesterase gene transcription levels of Loxostege sticticalis were studied using RT-PCR. Artificial diet HG was superior to artificial diet 12. These results provided a theoretical basis for further improvement of Loxostege sticticalis L. artificial feeding and feed formula.

  6. Structural and functional insights into Mimivirus ORFans

    Directory of Open Access Journals (Sweden)

    Fischer Daniel

    2007-05-01

    Full Text Available Abstract Background Mimivirus isolated from A. polyphaga is the largest virus discovered so far. It is unique among all the viruses in having genes related to translation, DNA repair and replication which bear close homology to eukaryotic genes. Nevertheless, only a small fraction of the proteins (33% encoded in this genome has been assigned a function. Furthermore, a large fraction of the unassigned protein sequences bear no sequence similarity to proteins from other genomes. These sequences are referred to as ORFans. Because of their lack of sequence similarity to other proteins, they can not be assigned putative functions using standard sequence comparison methods. As part of our genome-wide computational efforts aimed at characterizing Mimivirus ORFans, we have applied fold-recognition methods to predict the structure of these ORFans and further functions were derived based on conservation of functionally important residues in sequence-template alignments. Results Using fold recognition, we have identified highly confident computational 3D structural assignments for 21 Mimivirus ORFans. In addition, highly confident functional predictions for 6 of these ORFans were derived by analyzing the conservation of functional motifs between the predicted structures and proteins of known function. This analysis allowed us to classify these 6 previously unannotated ORFans into their specific protein families: carboxylesterase/thioesterase, metal-dependent deacetylase, P-loop kinases, 3-methyladenine DNA glycosylase, BTB domain and eukaryotic translation initiation factor eIF4E. Conclusion Using stringent fold recognition criteria we have assigned three-dimensional structures for 21 of the ORFans encoded in the Mimivirus genome. Further, based on the 3D models and an analysis of the conservation of functionally important residues and motifs, we were able to derive functional attributes for 6 of the ORFans. Our computational identification of important

  7. Selective inhibition of butyrylcholinesterase in vivo in horses by the feed-through larvacide Equitrol.

    Science.gov (United States)

    Karanth, Subramanya; Holbrook, Todd; MacAllister, Charles; Pope, Carey N

    2008-03-01

    ). Neither erythrocyte cholinesterase activity nor plasma carboxylesterase activity was affected by larvacide treatment in vivo. Muscle cholinesterase activity was highly variable among individual horses (pre-treatment range: 0.50-4.92nmole/min/mg protein), but there was no suggestion of a treatment-related reduction in muscle cholinesterase activity. These in vivo results confirm our previous in vitro studies indicating marked differential sensitivity of horse plasma and erythrocyte cholinesterase to inhibition by TCVP. Furthermore, the results suggest that recommended dosing levels of the TCVP-containing larvacide in horses are unlikely to affect acetylcholinesterase activities or disrupt cholinergic neurotransmission in target tissues.

  8. Invasive mechanism and management strategy of Bemisia tabaci(Gennadius) biotype B:Progress report of 973 Program on invasive alien species in China

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Bemisia tabaci(Gennadius) biotype B,called a "superbug",is one of the most harmful biotypes of this species complex worldwide.In this report,the invasive mechanism and management of B.tabaci biotype B,based on our 5-year studies,are presented.Six B.tabaci biotypes,B,Q,ZHJ1,ZHJ2,ZHJ3 and FJ1,have been identified in China.Biotype B dominates the other biotypes in many regions of the country.Genetic diversity in biotype B might be induced by host plant,geographical conditions,and/or insecticidal application.The activities of CarE(carboxylesterase) and GSTs(glutathione-S-transferase) in biotype B reared on cucumber and squash were greater than on other host plants,which might have increased its resistance to insecticides.The higher activities of detoxification enzymes in biotype B might be induced by the secondary metabolites in host plants.Higher adaptive ability of biotype B adults to adverse conditions might be linked to the expression of heat shock protein genes.The indigenous B.tabaci biotypes were displaced by the biotype B within 225 d.The asymmetric mating interactions and mutualism between biotype B and begomoviruses via its host plants speed up widespread invasion and displacement of other biotypes.B.tabaci biotype B displaced Trialeurodes vaporariorum(Westwood) after 4-7 generations under glasshouse conditions.Greater adaptive ability of the biotype B to adverse conditions and its rapid population increase might be the reasons of its successful displacement of T.vaporariorum.Greater ability of the biotype B to switch to different host plants may enrich its host plants,which might enable it to better compete with T.vaporariorum.Native predatory natural enemies possess greater ability to suppress B.tabaci under field conditions.The kairomones in the 3rd and 4th instars of biotype B may provide an important stimulus in host searching and location by its parasitoids.The present results provide useful information in explaining the mechanisms of genetic diversity

  9. Invasive mechanism and management strategy of Bemisia tabaci (Gennadius) biotype B: progress report of 973 Program on invasive alien species in China.

    Science.gov (United States)

    Wan, FangHao; Zhang, GuiFen; Liu, ShuSheng; Luo, Chen; Chu, Dong; Zhang, YouJun; Zang, LianSheng; Jiu, Min; Lü, ZhiChuang; Cui, XuHong; Zhang, LiPing; Zhang, Fan; Zhang, QingWen; Liu, WanXue; Liang, Pei; Lei, ZhongRen; Zhang, YongJun

    2009-01-01

    Bemisia tabaci (Gennadius) biotype B, called a "superbug", is one of the most harmful biotypes of this species complex worldwide. In this report, the invasive mechanism and management of B. tabaci biotype B, based on our 5-year studies, are presented. Six B. tabaci biotypes, B, Q, ZHJ1, ZHJ2, ZHJ3 and FJ1, have been identified in China. Biotype B dominates the other biotypes in many regions of the country. Genetic diversity in biotype B might be induced by host plant, geographical conditions, and/or insecticidal application. The activities of CarE (carboxylesterase) and GSTs (glutathione-S-transferase) in biotype B reared on cucumber and squash were greater than on other host plants, which might have increased its resistance to insecticides. The higher activities of detoxification enzymes in biotype B might be induced by the secondary metabolites in host plants. Higher adaptive ability of biotype B adults to adverse conditions might be linked to the expression of heat shock protein genes. The indigenous B. tabaci biotypes were displaced by the biotype B within 225 d. The asymmetric mating interactions and mutualism between biotype B and begomoviruses via its host plants speed up widespread invasion and displacement of other biotypes. B. tabaci biotype B displaced Trialeurodes vaporariorum (Westwood) after 4-7 generations under glasshouse conditions. Greater adaptive ability of the biotype B to adverse conditions and its rapid population increase might be the reasons of its successful displacement of T. vaporariorum. Greater ability of the biotype B to switch to different host plants may enrich its host plants, which might enable it to better compete with T. vaporariorum. Native predatory natural enemies possess greater ability to suppress B. tabaci under field conditions. The kairomones in the 3rd and 4th instars of biotype B may provide an important stimulus in host searching and location by its parasitoids. The present results provide useful information in

  10. Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.

    Directory of Open Access Journals (Sweden)

    Renato A Carvalho

    Full Text Available The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain or lambda-cyhalothrin (PYR strain were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE and pyrethroid (voltage-gated sodium channel, VGSC target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS. These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results

  11. Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity.

    Science.gov (United States)

    Robles-Mendoza, Cecilia; Zúñiga-Lagunes, Sebastian R; Ponce de León-Hill, Claudia A; Hernández-Soto, Jesús; Vanegas-Pérez, Cecilia

    2011-10-01

    The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (≈ 50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the

  12. Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes

    Directory of Open Access Journals (Sweden)

    Zhou Xuguo

    2009-10-01

    Full Text Available Abstract Background Termite lignocellulose digestion is achieved through a collaboration of host plus prokaryotic and eukaryotic symbionts. In the present work, we took a combined host and symbiont metatranscriptomic approach for investigating the digestive contributions of host and symbiont in the lower termite Reticulitermes flavipes. Our approach consisted of parallel high-throughput sequencing from (i a host gut cDNA library and (ii a hindgut symbiont cDNA library. Subsequently, we undertook functional analyses of newly identified phenoloxidases with potential importance as pretreatment enzymes in industrial lignocellulose processing. Results Over 10,000 expressed sequence tags (ESTs were sequenced from the 2 libraries that aligned into 6,555 putative transcripts, including 171 putative lignocellulase genes. Sequence analyses provided insights in two areas. First, a non-overlapping complement of host and symbiont (prokaryotic plus protist glycohydrolase gene families known to participate in cellulose, hemicellulose, alpha carbohydrate, and chitin degradation were identified. Of these, cellulases are contributed by host plus symbiont genomes, whereas hemicellulases are contributed exclusively by symbiont genomes. Second, a diverse complement of previously unknown genes that encode proteins with homology to lignase, antioxidant, and detoxification enzymes were identified exclusively from the host library (laccase, catalase, peroxidase, superoxide dismutase, carboxylesterase, cytochrome P450. Subsequently, functional analyses of phenoloxidase activity provided results that were strongly consistent with patterns of laccase gene expression. In particular, phenoloxidase activity and laccase gene expression are mostly restricted to symbiont-free foregut plus salivary gland tissues, and phenoloxidase activity is inducible by lignin feeding. Conclusion To our knowledge, this is the first time that a dual host-symbiont transcriptome sequencing effort

  13. Are deep-sea organisms dwelling within a submarine canyon more at risk from anthropogenic contamination than those from the adjacent open slope? A case study of Blanes canyon (NW Mediterranean)

    Science.gov (United States)

    Koenig, Samuel; Fernández, Pilar; Company, Joan B.; Huertas, David; Solé, Montserrat

    2013-11-01

    Due to their geomorphological structure and proximity to the coastline, submarine canyons may act as natural conduit routes for anthropogenic contaminants that are transported from surface waters to the deep-sea. Organisms dwelling in these canyon environments might thus be at risk of experiencing adverse health effects due to higher pollution exposure. To address this question, chemical and biochemical analyses were conducted on two of the most abundant deep-sea fish species in the study area, namely Alepocephalus rostratus and Lepidion lepidion, and the most abundant deep-sea commercial decapod crustacean Aristeus antennatus sampled inside Blanes canyon (BC) and on the adjacent open slope (OS). Persistent organic pollutants (POPs) levels, including polychlorinated biphenyl (PCB), dichlorodiphenyltrichloroethane (DDT) and derivatives, hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB) were determined in muscle tissue of selected samples from 900 m and 1500 m depth. Potential effects resulting from contaminant exposure were determined using hepatic biomarkers such as ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-deethylase (PROD), catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase (GST), total glutathione peroxidase (GPX), glutathione reductase (GR) and superoxide-dismutase (SOD) enzyme activities and lipid peroxidation levels (LP). L. lepidion and A. antennatus tissues exhibited higher POP levels inside BC compared to the OS at 900 m depth. These findings were consistent with biomarker data (i.e. enzymatic response to presence of contaminant agents). Elevated xenobiotic-metabolizing (EROD and PROD) and antioxidant enzymes (CAT and GPX) indicated higher contaminant exposure in both species caught within BC. No difference in POP accumulation between sites was observed in L. lepidion at 1500 m depth, nor in biomarker data, suggesting that the pollution gradient was less pronounced at greater depths. This trend was further corroborated

  14. [{sup 18}F]FE-SUPPY and [{sup 18}F]FE-SUPPY:2 - metabolic considerations

    Energy Technology Data Exchange (ETDEWEB)

    Haeusler, Daniela [Dept. of Nuclear Medicine, Medical Univ. of Vienna, A-1090 Vienna (Austria); Dept. of Pharmaceutical Technology and Biopharmaceutics, Univ. of Vienna, A-1090 Vienna (Austria); Nics, Lukas [Dept. of Nuclear Medicine, Medical Univ. of Vienna, A-1090 Vienna (Austria); Dept. of Nutritional Sciences, Univ. of Vienna, A-1090 Vienna (Austria); Mien, Leonhard-Key [Dept. of Nuclear Medicine, Medical Univ. of Vienna, A-1090 Vienna (Austria); Dept. of Pharmaceutical Technology and Biopharmaceutics, Univ. of Vienna, A-1090 Vienna (Austria); Ungersboeck, Johanna [Dept. of Nuclear Medicine, Medical Univ. of Vienna, A-1090 Vienna (Austria); Dept. of Inorganic Chemistry, Univ. of Vienna, A-1090 Vienna (Austria); Lanzenberger, Rupert R. [Dept. of Psychiatry and Psychotherapy, Medical Univ. of Vienna, A-1090 Vienna (Austria); Shanab, Karem [Dept. of Drug and Natural Product Synthesis, Univ. of Vienna, A-1090 Vienna (Austria); Sindelar, Karoline M. [Dept. of Nuclear Medicine, Medical Univ. of Vienna, A-1090 Vienna (Austria); Viernstein, Helmut [Dept. of Pharmaceutical Technology and Biopharmaceutics, Univ. of Vienna, A-1090 Vienna (Austria); Wagner, Karl-Heinz [Dept. of Nutritional Sciences, Univ. of Vienna, A-1090 Vienna (Austria); Dudczak, Robert; Kletter, Kurt [Dept. of Nuclear Medicine, Medical Univ. of Vienna, A-1090 Vienna (Austria); Wadsak, Wolfgang [Dept. of Nuclear Medicine, Medical Univ. of Vienna, A-1090 Vienna (Austria); Dept. of Inorganic Chemistry, Univ. of Vienna, A-1090 Vienna (Austria); Mitterhauser, Markus [Dept. of Nuclear Medicine, Medical Univ. of Vienna, A-1090 Vienna (Austria); Dept. of Pharmaceutical Technology and Biopharmaceutics, Univ. of Vienna, A-1090 Vienna (Austria); Hospital Pharmacy of the General Hospital of Vienna, A-1090 Vienna (Austria)], E-mail: markus.mitterhauser@meduniwien.ac.at

    2010-05-15

    Introduction: Recently, [{sup 18}F]FE-SUPPY and [{sup 18}F]FE-SUPPY:2 were introduced as the first positron emission tomography (PET) tracers for the adenosine A{sub 3} receptor. Thus, aim of the present study was the metabolic characterization of the two adenosine A{sub 3} receptor PET tracers. Methods: In vitro carboxylesterase (CES) experiments were conducted using incubation mixtures containing different concentrations of the two substrates, porcine CES and phosphate-buffered saline. Enzymatic reactions were stopped by adding acetonitrile/methanol (10:1) after various time points and analyzed by a high-performance liquid chromatography (HPLC) standard protocol. In vivo experiments were conducted in male wild-type rats; tracers were injected through a tail vein. Rats were sacrificed after various time points (n=3), and blood and brain samples were collected. Sample cleanup was performed by an HPLC standard protocol. Results: The rate of enzymatic hydrolysis by CES demonstrated Michaelis-Menten constants in a micromolar range (FE-SUPPY, 20.15 {mu}M, and FE-SUPPY:2, 13.11 {mu}M) and limiting velocities of 0.035 and 0.015 {mu}M/min for FE-SUPPY and FE-SUPPY:2, respectively. Degree of metabolism in blood showed the following: 15 min pi 47.7% of [{sup 18}F]FE-SUPPY was intact compared to 33.1% of [{sup 18}F]FE-SUPPY:2; 30 min pi 30.3% intact [{sup 18}F]FE-SUPPY was found compared to 15.6% [{sup 18}F]FE-SUPPY:2. In brain, [{sup 18}F]FE-SUPPY:2 formed an early hydrophilic metabolite, whereas metabolism of [{sup 18}F]FE-SUPPY was not observed before 30 min pi Conclusion: Knowing that metabolism in rats is several times faster than in human, we conclude that [{sup 18}F]FE-SUPPY should be stable for the typical time span of a clinical investigation. As a consequence, from a metabolic point of view, one would tend to decide in favor of [{sup 18}F]FE-SUPPY.

  15. The use of juvenile Solea solea as sentinel in the marine platform of the Ebre Delta: in vitro interaction of emerging contaminants with the liver detoxification system.

    Science.gov (United States)

    Crespo, Marina; Solé, Montserrat

    2016-10-01

    Juveniles of Solea solea were sampled during the spring season in three consecutive years at a marine site by the mouth of the Ebre river. The aim was to assess if the extractive works from the toxic load upstream the river could be reflected on the health status of the fish living at the immediate sea. The biomarkers selected for the in vivo field study are commonly used as indicators of chemical exposures. They include activities of energy metabolism: lactate dehydrogenase (LDH) and citrate synthase (CS); neurotoxicity: cholinesterases (ChE); xenobiotic metabolism: cytochrome P450 (CYP)-dependent: EROD and BFCOD, carboxylesterase (CbE), glutathione S-transferase (GST) and uridine diphosphate glucuronyltransferase (UDPGT); and oxidative stress parameters such as catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX) as well as levels of lipid peroxidation (LPO). These biomarkers were mostly analysed in liver but also in gills and muscle depending on their particular tissue distribution and role. A complementary in vitro approach was also sought to see the capacity of common emerging contaminants (pharmaceuticals and personal care products; PPCPs) to interact with the liver microsomal detoxification system of the fish (EROD, BFCOD and CbE activities). The results indicated that in fish sampled in 2015 there was an enhancement in detoxification parameters (EROD, BFCOD and gill GR), muscular ChEs and gill CS, but a decrease in CbE activity and a marked oxidative stress situation (increased LPO and decreased CAT activity). Also, 4 out of the 10 PPCPs tested in vitro were able to interact with the CYP3A4 (BFCOD) enzymatic system while the lipid regulators simvastatin and fenofibrate inhibited CbE activity, as it occurs in higher vertebrates. The in vivo results support the use of a multibiomarker approach when assessing the disturbances due to chemical exposures, not only spatially but also over time, once the influence of other variables has been

  16. Insecticidal Activity of Melaleuca alternifolia Essential Oil and RNA-Seq Analysis of Sitophilus zeamais Transcriptome in Response to Oil Fumigation

    Science.gov (United States)

    Liao, Min; Xiao, Jin-Jing; Zhou, Li-Jun; Liu, Yang; Wu, Xiang-Wei; Hua, Ri-Mao; Wang, Gui-Rong; Cao, Hai-Qun

    2016-01-01

    Background The cereal weevil, Sitophilus zeamais is one of the most destructive pests of stored cereals worldwide. Frequent use of fumigants for managing stored-product insects has led to the development of resistance in insects. Essential oils from aromatic plants including the tea oil plant, Melaleuca alternifolia may provide environmentally friendly alternatives to currently used pest control agents. However, little is known about molecular events involved in stored-product insects in response to plant essential oil fumigation. Results M. alternifolia essential oil was shown to possess the fumigant toxicity against S. zeamais. The constituent, terpinen-4-ol was the most effective compound for fumigant toxicity. M. alternifolia essential oil significantly inhibited the activity of three enzymes in S. zeamais, including two detoxifying enzymes, glutathione S-transferase (GST), and carboxylesterase (CarE), as well as a nerve conduction enzyme, acetylcholinesterase (AChE). Comparative transcriptome analysis of S. zeamais through RNA-Seq identified a total of 3,562 differentially expressed genes (DEGs), of which 2,836 and 726 were up-regulated and down-regulated in response to M. alternifolia essential oil fumigation, respectively. Based on gene ontology (GO) analysis, the majority of DEGs were involved in insecticide detoxification and mitochondrial function. Furthermore, an abundance of DEGs mapped into the metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database were associated with respiration and metabolism of xenobiotics, including cytochrome P450s, CarEs, GSTs, and ATP-binding cassette transporters (ABC transporters). Some DEGs mapped into the proteasome and phagosome pathway were found to be significantly enriched. These results led us to propose a model of insecticide action that M. alternifolia essential oil likely directly affects the hydrogen carrier to block the electron flow and interfere energy synthesis in

  17. Rhodnius prolixus supergene families of enzymes potentially associated with insecticide resistance.

    Science.gov (United States)

    Schama, Renata; Pedrini, Nicolás; Juárez, M Patricia; Nelson, David R; Torres, André Q; Valle, Denise; Mesquita, Rafael D

    2016-02-01

    Chagas disease or American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi. Once known as an endemic health problem of poor rural populations in Latin American countries, it has now spread worldwide. The parasite is transmitted by triatomine bugs, of which Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae) is one of the vectors and a model organism. This species occurs mainly in Central and South American countries where the disease is endemic. Disease prevention focuses on vector control programs that, in general, rely intensely on insecticide use. However, the massive use of chemical insecticides can lead to resistance. One of the major mechanisms is known as metabolic resistance that is associated with an increase in the expression or activity of detoxification genes. Three of the enzyme families that are involved in this process - carboxylesterases (CCE), glutathione s-transferases (GST) and cytochrome P450s (CYP) - are analyzed in the R. prolixus genome. A similar set of detoxification genes to those of the Hemipteran Acyrthosiphon pisum but smaller than in most dipteran species was found in R. prolixus genome. All major CCE classes (43 genes found) are present but the pheromone/hormone processing class had fewer genes than usual. One main expansion was detected on the detoxification/dietary class. The phosphotriesterase family, recently associated with insecticide resistance, was also represented with one gene. One microsomal GST gene was found and the cytosolic GST gene count (14 genes) is extremely low when compared to the other hemipteran species with sequenced genomes. However, this is similar to Apis mellifera, a species known for its deficit in detoxification genes. In R. prolixus 88 CYP genes were found, with representatives in the four clans (CYP2, CYP3, CYP4 and mitochondrial) usually found in insects. R. prolixus seems to have smaller species-specific expansions of CYP genes than

  18. Biologically Based Methods for Control of Fumonisin-Producing Fusarium Species and Reduction of the Fumonisins.

    Science.gov (United States)

    Alberts, Johanna F; van Zyl, Willem H; Gelderblom, Wentzel C A

    2016-01-01

    Infection by the fumonisin-producing Fusarium spp. and subsequent fumonisin contamination of maize adversely affect international trade and economy with deleterious effects on human and animal health. In developed countries high standards of the major food suppliers and retailers are upheld and regulatory controls deter the importation and local marketing of fumonisin-contaminated food products. In developing countries regulatory measures are either lacking or poorly enforced, due to food insecurity, resulting in an increased mycotoxin exposure. The lack and poor accessibility of effective and environmentally safe control methods have led to an increased interest in practical and biological alternatives to reduce fumonisin intake. These include the application of natural resources, including plants, microbial cultures, genetic material thereof, or clay minerals pre- and post-harvest. Pre-harvest approaches include breeding for resistant maize cultivars, introduction of biocontrol microorganisms, application of phenolic plant extracts, and expression of antifungal proteins and fumonisin degrading enzymes in transgenic maize cultivars. Post-harvest approaches include the removal of fumonisins by natural clay adsorbents and enzymatic degradation of fumonisins through decarboxylation and deamination by recombinant carboxylesterase and aminotransferase enzymes. Although, the knowledge base on biological control methods has expanded, only a limited number of authorized decontamination products and methods are commercially available. As many studies detailed the use of natural compounds in vitro, concepts in reducing fumonisin contamination should be developed further for application in planta and in the field pre-harvest, post-harvest, and during storage and food-processing. In developed countries an integrated approach, involving good agricultural management practices, hazard analysis and critical control point (HACCP) production, and storage management, together with

  19. Biologically Based Methods for Control of Fumonisin-producing Fusarium species and Reduction of the Fumonisins

    Directory of Open Access Journals (Sweden)

    Johanna Francina Alberts

    2016-04-01

    Full Text Available Infection by the fumonisin-producing Fusarium spp. and subsequent fumonisin contamination of maize adversely affect international trade and economy with deleterious effects on human and animal health. In developed countries high standards of the major food suppliers and retailers are upheld and regulatory controls deter the importation and local marketing of fumonisin-contaminated food products. In developing countries regulatory measures are either lacking or poorly enforced, due to food insecurity, resulting in an increased mycotoxin exposure. The lack and poor accessibility of effective and environmentally safe control methods have led to an increased interest in practical and biological alternatives to reduce fumonisin intake. These include the application of natural resources, including plants, microbial cultures, genetic material thereof or clay minerals pre- and postharvest. Pre-harvest approaches include breeding for resistant maize cultivars, introduction of biocontrol microorganisms, application of phenolic plant extracts, and expression of antifungal proteins and fumonisin degrading enzymes in transgenic maize cultivars. Postharvest approaches include the removal of fumonisins by natural clay adsorbents and enzymatic degradation of fumonisins through decarboxylation and deamination by recombinant carboxylesterase and aminotransferase enzymes. Although the knowledge base on biological control methods has expanded, only a limited number of authorized decontamination products and methods are commercially available. As many studies detailed the use of natural compounds in vitro, concepts in reducing fumonisin contamination should be developed further for application in planta and in the field pre-harvest, postharvest, and during storage and food-processing. In developed countries an integrated approach, involving good agricultural management practices, hazard analysis and critical control point (HACCP production and storage management

  20. Combined Transcriptomic and Proteomic Analysis of the Posterior Salivary Gland from the Southern Blue-Ringed Octopus and the Southern Sand Octopus.

    Science.gov (United States)

    Whitelaw, Brooke L; Strugnell, Jan M; Faou, Pierre; da Fonseca, Rute R; Hall, Nathan E; Norman, Mark; Finn, Julian; Cooke, Ira R

    2016-09-02

    This study provides comprehensive proteomic profiles from the venom producing posterior salivary glands of octopus (superorder Octopodiformes) species. A combined transcriptomic and proteomic approach was used to identify 1703 proteins from the posterior salivary gland of the southern blue-ringed octopus, Hapalochlaena maculosa and 1300 proteins from the posterior salivary gland of the southern sand octopus, Octopus kaurna. The two proteomes were broadly similar; clustering of proteins into orthogroups revealed 937 that were shared between species. Serine proteases were particularly diverse and abundant in both species. Other abundant proteins included a large number of secreted proteins, many of which had no known conserved domains, or homology to proteins with known function. On the basis of homology to known venom proteins, 23 putative toxins were identified in H. maculosa and 24 in O. kaurna. These toxins span nine protein families: CAP (cysteine rich secretory proteins, antigen 5, parthenogenesis related), chitinase, carboxylesterase, DNase, hyaluronidase, metalloprotease, phospholipase, serine protease and tachykinin. Serine proteases were responsible for 70.9% and 86.3% of putative toxin expression in H. maculosa and O. kaurna, respectively, as determined using intensity based absolute quantification (iBAQ) measurements. Phylogenetic analysis of the putative toxin serine proteases revealed a similar suite of diverse proteins present in both species. Posterior salivary gland composition of H. maculosa and O. kaurna differ in several key aspects. While O. kaurna expressed the proteinaceous neurotoxin, tachykinin, this was absent from H. maculosa, perhaps reflecting the acquisition of a potent nonproteinaceous neurotoxin, tetrodotoxin (TTX) produced by bacteria in the salivary glands of that species. The dispersal factor, hyaluronidase was particularly abundant in H. maculosa. Chitinase was abundant in both species and is believed to facilitate

  1. An enhanced in vivo stable isotope labeling by amino acids in cell culture (SILAC) model for quantification of drug metabolism enzymes.

    Science.gov (United States)

    MacLeod, A Kenneth; Fallon, Padraic G; Sharp, Sheila; Henderson, Colin J; Wolf, C Roland; Huang, Jeffrey T-J

    2015-03-01

    Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of

  2. Low concentrations of metal mixture exposures have adverse effects on selected biomarkers of Xenopus laevis tadpoles

    Energy Technology Data Exchange (ETDEWEB)

    Yologlu, Ertan, E-mail: ertanyologlu82@gmail.com [Adiyaman University, Faculty of Education, Department of Science Education, 02040 Adiyaman (Turkey); Ozmen, Murat [Inonu University, Laboratory of Environmental Toxicology, Department of Biology, Faculty of Arts & Science, 44280 Malatya (Turkey)

    2015-11-15

    Highlights: • Selected metal mixtures were evaluated for toxicity of safety limit concentrations. • Xenopus laevis tadpoles were used as model test organism. • Combinations of LC{sub 50} and LC{sub 50}/2 caused 100% lethality for some metals. • Metals did not change metallothionein levels in low concentrations. • Selected enzyme activities showed induction after low concentration exposures. - Abstract: Polluted ecosystems may contain mixtures of metals, such that the combinations of metals, even in low concentrations, may cause adverse effects. In the present study, we focused on toxic effects of mixtures of selected metals, the LC{sub 50} values, and also their safety limit in aquatic systems imposed by the European legislation using a model organism. Xenopus laevis tadpoles were used as test organisms. They were exposed to metals or their combinations due to 96-h LC{sub 50} values. Glutathione S-transferase (GST), glutathione reductase (GR), acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione peroxidase (GPx), and catalase (CAT) levels were evaluated. Metallothionein concentrations were also determined. The LC{sub 50}s for Cd, Pb, and Cu were calculated as 5.81 mg AI/L, 123.05 mg AI/L, and 0.85 mg AI/L, respectively. Low lethality ratios were observed with unary exposure of each metal in lower concentrations. Double or triple combinations of LC{sub 50} and LC{sub 50}/2 concentrations caused 100% lethality with Cd + Cu and Pb + Cd + Cu mixtures, while the Pb + Cu mixture also caused high lethal ratios. The selected enzyme activities were significantly affected by metals or mixtures, and dose-related effects were determined. The metallothionein levels generally increased as related to concentration in unary metals and mixtures. Acceptable limit values of unary metals and mixtures did not significantly change metallothionein levels. The results suggest that oxidative stress-related mechanisms are involved in the toxicity induced by selected

  3. Pharmacokinetic drug interactions with clopidogrel: updated review and risk management in combination therapy

    Directory of Open Access Journals (Sweden)

    Wang ZY

    2015-03-01

    inhibitors on clopidogrel (omeprazole, esomeprazole versus pantoprazole, rabeprazole, the effects of rifampicin on clopidogrel versus ticagrelor and prasugrel, and the effects of calcium channel blockers on clopidogrel (amlodipine versus P-glycoprotein-inhibiting calcium channel blockers. The mechanism of the DDIs with clopidogrel involves modulating CYP enzymes (eg, CYP2B6, CYP2C8, CYP2C19, and CYP3A4, paraoxonase-1, hepatic carboxylesterase 1, P-glycoprotein, and organic anion transporter family member 1B1.Conclusion: Effective and safe clopidogrel combination therapy can be achieved by increasing the awareness of potential changes in efficacy and toxicity, rationally selecting alternatives, tailoring drug therapy based on genotype, checking the appropriateness of physician orders, and performing therapeutic monitoring. Keywords: clopidogrel, drug–drug interactions, drug metabolism, drug transporter, genotype, pharmacokinetics, polypharmacy, pharmacogenetics, P2Y12 receptor inhibitors, risk management 

  4. 昆虫对拟除虫菊酯类杀虫剂的代谢抗性机制研究进展%Mechanism of insect metabolic resistance to pyrethroid insecticides

    Institute of Scientific and Technical Information of China (English)

    陈澄宇; 史雪岩; 髙希武

    2016-01-01

    insects detoxification enzymes such as cytochrome P450, carboxylesterase and glutathioneS-transferase resulted in the metabolic resistance of insects to pyrethroid insecticide. Especially the changes of the activity and relative gene expression level of these detoxification enzymes are the main mechanisms of insects metabolic resistance to pyrethroids. Clarifying the metabolic resistance mechanisms of resistant insects are beneficial to rational use of pyrethroids insecticides and delay the development of insects resistance to pyrethroids. On the basis of summarizing the metabolic pathways of pyrethroids and the related metabolic enzymes, this article provides an overview of the new achievements on mechanisms of insect metabolic resistance to pyrethroid insecticides.

  5. Comparison of esterase isozymes in two color varieties of Tenebrio molitor L.%2种色型黄粉虫酯酶同工酶的比较

    Institute of Scientific and Technical Information of China (English)

    黄琼; 胡杰; 苟琳

    2011-01-01

    only at this stage. The enzymatic activities of carboxylesterase ( CarE), acetylcholine esterase ( AchE), acid phosphatase ( ACP) and alkaline phosphatase (AKP) in black-color variety were always higher than those in yellow-color variety at all stages from egg to adult. While in the same variety, The enzymatic activity of CarE, AchE, ACP and AKP varied in the order of egg stage < adult stage < pupal stage < larval stage. The results above build up some underlying data for further studying the insecticide resistance and variety selection of the mealworm beetles.

  6. Variations in susceptibility to common insecticides and resistance mechanisms among morphologically identified sibling species of the malaria vector Anopheles subpictus in Sri Lanka

    Directory of Open Access Journals (Sweden)

    Surendran Sinnathamby N

    2012-02-01

    DDT resistance than species B. Malathion resistance in both species C and D may be caused by elevated GST activity and an altered insensitive target site in AChE. In addition, a carboxylesterase based malathion resistance mechanisms was also detected in species C and D. Elevated esterase levels in species C and D might have contributed to the low levels of pyrethroid resistance. However an absence of elevated activity of monooxygenases in species B, C and D indicates that monooxygenases are unlikely to be the cause of this partial resistance to pyrethroids. Conclusions The differences in insecticide susceptibility and insecticide resistance mechanism shown by An. subpictus sibling species are important considerations for developing the malaria control and eradication program in Sri Lanka. Similar studies on species complexes of other anopheline vectors of malaria are necessary for effective malaria control worldwide. The differential susceptibility findings are also consistent with most, if not all, morphologically identified An. subpictus species B in Sri Lanka belonging to the An. sundaicus complex. There is a need therefore to develop molecular techniques that can be used to differentiate morphologically similar anopheline species in field conditions for more effective vector control.

  7. 多杀菌素对黄褐天幕毛虫解毒酶及保护酶的影响%Effect of Spinosad on the Detoxifying and Protective Enzymes of Malacosoma neustria testacea

    Institute of Scientific and Technical Information of China (English)

    刘丹; 严善春; 曹传旺; 廖月枝

    2012-01-01

    为了研究多杀菌素对黄褐天幕毛虫的杀虫活性及作用机制,采用叶片药膜法测定多杀菌素对黄褐天幕毛虫幼虫的毒杀效力,并用分光光度法检测LC50剂量药剂处理后不同时间点幼虫体内各种解毒酶和保护酶活性的变化.结果表明:多杀菌素对4龄、5龄幼虫均表现出极高的杀虫活性,见效速度块;而且黄褐天幕毛虫4龄、5龄幼虫经过多杀菌素处理后,谷胱甘肽S-转移酶均表现为复杂的抑制-激活-再抑制作用,多功能氧化酶活性则均表现为先抑制后激活的作用.多杀菌素对4龄幼虫羧酸酯酶活性具有激活作用,对乙酰胆碱酯酶和过氧化物酶均具有先激活后抑制作用,对超氧化物歧化酶具有先激活再抑制后激活的作用,对过氧化氢酶则表现为复杂的先抑制再激活后抑制作用.对5龄幼虫羧酸酯酶、乙酰胆碱酯酶、超氧化物歧化酶及过氧化物酶活性具有明显的抑制作用,而对过氧化氢酶则表现为先抑制再激活后抑制的作用.因此,多杀菌素能有效干扰昆虫解毒和保护系统,扰乱其正常的生理代谢,从而起到较好的毒杀效果.%To study the insecticidal activity and toxicity mechanism of spinosad, we assayed bioactivity of spinosad by leaf membrane method and its effects on the activities of detoxifying and protective enzymes in Malacosoma neustria testacea larvae by spectrophotometry. The results showed that spinosad had an extremely high toxicity against the 4' and 5' instar larvae. The glutathione S-transfer (GST) activity in 4th and 5th instar larvae was firstly inhibited, then induced, and finally inhibited, while the mixed-functional oxidase ( MFO) activity was inhibited and then enhanced. The induced effect on carboxylesterase ( CarE) , the induced and inhibited effect on acetylcholinesterase ( AchE) and peroxidase ( POD) , and the complicated effects on superoxide dismutase ( SOD) and Catalase ( CAT) were determined in 4

  8. Effect of low dose spinetoram on detoxification enzymes inPlutella xylostella%低剂量乙基多杀菌素对小菜蛾解毒酶的影响

    Institute of Scientific and Technical Information of China (English)

    田雪莲; 尹显慧; 龙友华; 李明; 蔡滔; 李荣玉; 朱流红

    2016-01-01

    为探讨低剂量乙基多杀菌素对小菜蛾Plutella xylostella (L.)解毒酶的影响,采用叶片浸渍法,测定了乙基多杀菌素和多杀菌素对小菜蛾敏感种群的毒力,并比较了低剂量(LC25和LC50)处理6、12、24、48和72 h时小菜蛾体内羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GST)和多功能氧化酶系(MFOs)活性的变化动态。结果表明:乙基多杀菌素对小菜蛾的杀虫活性优于多杀菌素,处理48 h后其 LC25和 LC50浓度分别为0.018和0.048 mg/L,经此低剂量浓度处理后,小菜蛾 CarE活性波动较大,6~24 h,处理组 CarE活性高于对照组,且均呈先升后降趋势,24~72 h,处理组 CarE活性均低于对照组,并且具有一定的时间效应;对 GST具有明显的诱导作用,GST活性均高于对照组;对 MFOs具有明显的抑制作用,除在48 h时相差不大外,其他时间 MFOs活性均显著低于对照组。结果表明,GST可能参与了乙基多杀菌素在小菜蛾体内的代谢。%To clarify the effects of low dose spinetoram on the activitiy of detoxifying enzymes in Plutella xylostella (L.), the toxicities of spinetoram and spinosad to susceptible population ofP. xylostella were assayed using leaf dipping method. And the changing dynamics of detoxifying enzymes of carboxylesterase, glutathioneS-transferase and mixed-functional oxidase were measured after exposure to LC25 and LC50 doses of spinetoram at 6, 12, 24, 48 and 72 h, respectively. The results showed that the spinetoram had higher toxicity toP. xylostella than that of spinosad. The 48 h LC25 and LC50 of spinetoram were 0.018 and 0.048 mg/L, respectively. After treatment with low concentrations spinetoram, a large fluctuation of the CarE was observed. CarE activity in the treatment strain were higher than those in the untreated control strain after treatment 6-24 h, and showed tendency of first increasing and then decreasing. Whereas, from 24 h to 72 h, CarE activity was lower

  9. Effects of Bt Transgenic Cotton on Occurrence of Cotton Spider Mites in Relation to the Secondary Metabolites in Cotton%Bt棉对棉叶螨发生的影响及与次生代谢物质的关系

    Institute of Scientific and Technical Information of China (English)

    马惠; 赵鸣; 夏晓明; 王红艳; 董合忠

    2012-01-01

    We sought to determine the effects of Sf transgenic cotton on occurrence of cotton spider mites, and the relationship with the secondary metabolites in cotton plants. Using four Bt cotton varieties and a non-Bf cotton, CCRI 12, as the control, the abundance and developmental duration of cotton spider mites, and also the activity of their carboxylesterase(CarE) were determined, as well as the gossypol and tannin levels in cotton leaves, both in the field and in the greenhouse. Cotton spider mites were more abundant on four Bt cotton varieties than on the common cotton variety CCRI 12. The development period of cotton spider mites fed on four Bt cotton varieties was shorter than on CCRI 12. The gossypol and tannin levels in four Bt cotton varieties were significantly lower than in CCRI 12, although these also varied greatly among the Bt cotton varieties. The activity of CarE in mites fed on CCRI 12 was significantly lower than in those fed on the four Bt varieties. In general it appears that changes in secondary metabolite levels affect the occurrence of cotton spider mites in Bt cotton.%为明确Bt棉对棉叶螨发生的影响及其与棉株次生代谢物质的关系,以4个Bt棉品种为材料,以不携带Bt基因的常规棉为对照,在大田和温室中调查了棉叶螨的发生趋势,室内观察了棉叶螨的发育历期,测定了不同品种棉花叶片的棉酚和单宁含量,以及取食不同棉花品种的棉叶螨羧酸酯酶的比活力.结果表明,棉叶螨在4个Bt棉品种上的发生显著重于非Bt棉中棉所12,取食4个Bt棉品种的棉叶螨发育历期均明显短于取食中棉所12的棉叶螨的发育历期.4个抗虫棉品种间棉酚和单宁含量虽有差异,但均显著低于中棉所12,而取食中棉所12的棉叶螨的羧酸酯酶比活力也显著低于4个Bt棉品种.Bt棉本身次生代谢物质含量的改变可能影响了棉叶螨的发生.

  10. Effects of Chlorpyrifos on Food Utilization and Detoxifying Enzymes and Acetylcholinesterase of Lymantria dispar%毒死蜱对舞毒蛾食物利用和解毒酶及AChE活性的影响

    Institute of Scientific and Technical Information of China (English)

    李慧; 严善春; 王志英; 葛士林; 曹传旺

    2011-01-01

    采用质量法和酶活性测定法研究了毒死蜱对舞毒蛾(Lymantria dispar)3龄幼虫食物利用的影响,并测定了其毒力及解毒酶、乙酰胆碱酯酶(AChE)的活性.用亚致死浓度(1.5 mg·L-1)毒死蜱处理小黑杨叶片,饲喂舞毒蛾3龄幼虫,其幼虫生长率(RGR)、食物利用率(ECI)和食物转化率(ECD)均显著低于对照,而近似消化率(AD)显著高于对照,相对取食量(RCR)处理和对照间差异不显著.毒死蜱对舞毒蛾幼虫24 h致死中浓度(LC50)为5.86 mg·L-1,其毒力低于三氟氯氰菊酯而高于氧化乐果.毒死蜱对舞毒蛾3龄幼虫体内羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)和AChE均有抑制作用,抑制程度为CarE>AChE>GSTs.毒死蜱通过影响舞毒蛾食物利用和抑制酶活性而表现出杀虫活性,为一种有效防治舞毒蛾的有机磷杀虫剂.%The effects of sublethal dose of chlorpyrifos (CPF) on food utilization of gypsy moth, Lymantria dispar,as well as CPF toxicity and enzyme activities of carboxylesterase ( CarE ), glutathione S-transferases (GSTs) and acetylcholinesterase (AChE) were evaluated in the 3rd-instar gypsy moth larvae using gravimetric method and measuring enzyme activities. Relative growth rate (RGR), efficiency of the conversion of ingested food (ECI) and efficiency of the conversion of digested food (ECD) of 3rd-instar larvae fed on poplar leaves treated by sublethal concentration of CPF were significantly lower than thosc fed on normal polar leaves. However, approximate digestibility (AD) of the treatment was significantly higher than that of control. The relative consumption rate (RCR) of the treatment and control groups were not significantly different. The 24 h LC50 of CPF to the gypsy moth 3rd-instar larvae was 5.86 mg · L-1, which was higher than that of omethoate but lower than that of cyhalothrin. In vitro inhibition assay indicated that the activities of AChE, CarE and GSTs were inhibited by CPF depended on concentrations

  11. Proteomic fingerprinting of N-linked glycoproteins involved in hepatocellular carcinoma%肝细胞癌中差异表达 N-连接糖蛋白的分析鉴定

    Institute of Scientific and Technical Information of China (English)

    马瑾; 齐义军; 刘瑞敏; 王明; 张天; 朱晗; 马远方

    2014-01-01

    目的:分析鉴定与肝细胞癌( HCC)发生发展相关的差异表达的N-连接糖蛋白。方法应用刀豆蛋白凝集素(ConA)、晶状体凝集素(LCH)和雪花凝集素(GNA)组成的亲合层析柱富集10对HCC和癌旁非癌组织中N-连接糖蛋白、二维电泳(2DE)比较分析差异表达的蛋白质点、串联质谱鉴定差异表达蛋白,Western blotting 验证人羧酸酯酶1(hCE1)、触珠蛋白(HP)及组织蛋白酶D(CD)的差异表达;体外侵袭实验检测CD表达沉默后对HCC的侵袭力影响。结果质谱、生物信息学等技术鉴定了28个与HCC相关的差异表达蛋白( HCC中高表达和低表达的蛋白均为14个),Western blotting验证hCE1、HP在HCC中明显低表达,组织蛋白酶D前体( pCD)在HCC中高表达;而ConA亲和层析柱富集的ConA-CD在HCC中显著高表达。 CD-siRNA介导的CD表达沉默能够显著降低肝癌细胞系SNU449、SNU473的体外侵袭能力。结论 HP、hCE1蛋白表达变化和CD N-糖链变异可能参与了HCC发生发展过程。%Objective To identify differentially expressed N-linked glycoproteins between hepatocellular carcinoma ( HCC) and adjacent non-tumorous liver tissues .Methods N-linked glycoproteome was extracted by multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), and snowdrop lectin (GNA) and subsequently subjected to two-dimensional electrophoresis ( 2DE ) and mass spectrometry ( MS ) for identification of differential glycoproteins between 10 pairs of HCC and adjacent non-cancer tissue .Western blotting was used to verify different expression of human liver carboxylesterase 1 (hCE1), haptoglobin (HP)and cathepsin D (CD).Invasion potential in vitro was examined after si-RNA mediated CD gene scilencing .Results LC-ESI-MS/MS identified a total of 28 differentially expressed glycoproteins (14 up-regulation and 14 down-regulated).Western blotting detected consistent down

  12. Rufinamide: clinical pharmacokinetics and concentration-response relationships in patients with epilepsy.

    Science.gov (United States)

    Perucca, Emilio; Cloyd, James; Critchley, David; Fuseau, Eliane

    2008-07-01

    Rufinamide is a new, orally active antiepileptic drug (AED), which has been found to be effective in the treatment of partial seizures and drop attacks associated with the Lennox-Gastaut syndrome. When taken with food, rufinamide is relatively well absorbed in the lower dose range, with approximately dose-proportional plasma concentrations up to 1,600 mg/day, but less than dose-proportional plasma concentrations at higher doses due to reduced oral bioavailability. Rufinamide is not extensively bound to plasma proteins. During repeated dosing, steady state is reached within 2 days, consistent with its elimination half-life of 6-10 h. The apparent volume of distribution (V(d)/F) and apparent oral clearance (CL/F) are related to body size, the best predictor being body surface area. Rufinamide is not a substrate of cytochrome P450 (CYP450) enzymes and is extensively metabolized via hydrolysis by carboxylesterases to a pharmacologically inactive carboxylic acid derivative, which is excreted in the urine. Rufinamide pharmacokinetics are not affected by impaired renal function. Potential differences in rufinamide pharmacokinetics between children and adults have not been investigated systematically in formal studies. Although population pharmacokinetic modeling suggests that in the absence of interacting comedication rufinamide CL/F may be higher in children than in adults, a meaningful comparison of data across age groups is complicated by age-related differences in doses and in proportion of patients receiving drugs known to increase or to decrease rufinamide CL/F. A study investigating the effect of rufinamide on the pharmacokinetics of the CYP3A4 substrate triazolam and an oral contraceptive interaction study showed that rufinamide has some enzyme-inducing potential in man. Findings from population pharmacokinetic modeling indicate that rufinamide does not modify the CL/F of topiramate or valproic acid, but may slightly increase the CL/F of carbamazepine and

  13. Rufinamide for pediatric patients with Lennox-Gastaut syndrome: a comprehensive overview.

    Science.gov (United States)

    Wier, Heather Ann; Cerna, Ana; So, Tsz-Yin

    2011-04-01

    Rufinamide is a triazole derivative with broad-spectrum antiepileptic effects that is unrelated to any antiepileptic drug currently on the market. The European Commission and the US FDA approved rufinamide in 2007 and 2008, respectively, for adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in children 4 years of age or older and adults. The mechanism of action of rufinamide is not completely understood but it is believed to prolong the inactive state of sodium channels, therefore limiting excessive firing of sodium-dependent action potentials. Rufinamide is well absorbed when taken with food, with an absolute bioavailability between 70% and 85%. The elimination half-life of the drug is around 6-10 hours, with a time to maximum plasma concentration (C(max)) of approximately 4-6 hours. The C(max) at a dosage of 10 mg/kg/day and 30 mg/kg/day is 4.01 μg/mL and 8.68 μg/mL, respectively, and the area under the plasma concentration-time curve from time 0 to 12 hours was 37.8 ± 47 μg · h/mL and 89.3 ± 58 μg · h/mL, respectively. Rufinamide exerts non-linear pharmacokinetics with increasing doses. The volume of distribution in children is similar to that in adults (0.8-1.2 L/kg) and the drug binds rather poorly to plasma protein (26.2-34.8%). Rufinamide is mainly metabolized by carboxylesterases to an inactive metabolite (CGP 47292), and the majority of the metabolites are excreted in the urine (91%). No dosage adjustment is required in patients with renal dysfunction. Rufinamide does not affect the plasma concentration of other antiepileptics, but phenytoin, phenobarbital, valproate, and primidone affect the clearance of rufinamide. In a clinical study of 138 patients averaging 12 years of age, rufinamide used as an adjunctive therapy (with an initial dosage of 10 mg/kg/day up to a target dosage of 45 mg/kg/day) in patients with Lennox-Gastaut syndrome reduced the median total seizure frequency by 32

  14. Plasma B-esterase activities in European raptors.

    Science.gov (United States)

    Roy, Claudie; Grolleau, Gérard; Chamoulaud, Serge; Rivière, Jean-Louis

    2005-01-01

    B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and

  15. 抗吡虫啉棉蚜对其他新烟碱类药剂的交互抗性及相关酶的活性变化%Cross-resistance of the imidacloprid-resistant population of Aphis gossypii Glover (Homoptera: Aphididae ) to other neonicotinoid insecticides and changes in activities of related enzymes

    Institute of Scientific and Technical Information of China (English)

    史晓斌; 石绪根; 王红艳; 夏晓明; 王开运

    2011-01-01

    In order to clarify the cross-resistance and the change in related enzymes of the imidacloprid-resistant population of Aphis gossypii to other neonicotinoid insecticides, the bioassay method was used to determine the cross-resistance of different resistant populations of imidacloprid-resistant A. Gossypii to dinotefuran and nitenpyram, and the synergism of three detoxification enzyme inhibitors to imidacloprid and other two neonicotinoid insecticides using cotton aphids of the imidacloprid-resistant population selected in the laboratory, the Xiajin resistant population in the field in Shandong and the susceptible population. The activities of detoxification enzymes and AChE of three populations of cotton aphid and the inhibition effect of insecticides were determined through biochemical analysis. The results showed that the imidacloprid-resistant population and the Xiajin resistant population exhibited no cross-resistance to dinotefuran, but showed 5. 28-fold and 4. 89-fold cross-resistance to nitenpyram, respectively. Dinotefuran could significantly inhibit the activities of CarE, GST and AChE of the imidacloprid-resistant cotton aphid. Nitenpyram showed little effect on the activities of CarE, GST and AChE of the imidacloprid-resistant cotton aphid. Carboxylesterase inhibitor TPP and mixed-functional oxidase inhibitor PBO had obvious synergism to imidacloprid and nitenpyram, while had little synergism to dinotefuran. Glutathione-S-transferase inhibitor DEM showed no obvious synergism to the three insecticides. Dinotefuran and nitenpyram could inhibit the activities of detoxification enzymes and AChE of the imidacloprid-resistant conton aphid, with dinotefuran showing significant effect. The results demonstrate the great application value of dinotefuran in control of the imidacloprid-resistant cotton aphid, and its structure can provide a reference to the development of neonicotinoid insecticides in the future.%为明确抗吡虫啉棉蚜Aphis gossypii对其他

  16. Synergisms and Physiological and Biochemical Mechanisms of Three New Mixtures against Pieris rapae L.%3种新型复配剂对菜青虫的增效作用及生理生化机制

    Institute of Scientific and Technical Information of China (English)

    米凤玉; 王永模; ELTAYEB E Mansour; 张国安

    2011-01-01

    [方法]为明确乙酰甲胺磷·噻虫嗪水乳剂、水分散粒剂和毒死蜱·噻虫嗪水分散粒剂3种新型复配剂的增效作用及增效机理,室内采用喷雾法测定了对菜青虫的增效作用;通过生化测定法研究了其对菜青虫体内羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)和乙酰胆碱酯酶(AChE)活力的影响;采用石蜡切片显微观察法分析了3种复配剂处理后菜青虫中肠组织细胞的变化.[结果]3种复配剂对菜青虫都有很好的杀虫活性,处理48 h后的共毒系数分别为13110、459.04、204.94,都表现为明显的增效作用;乙酰甲胺磷·噻虫嗪水乳剂显著抑制CarE、GSTs和AChE活力,抑制率分别为90.04%、74.98%、71.59%;乙酰甲胺磷·噻虫嗪水分散粒剂对3种酶活力的抑制率分别为72.11%、74.98%、90.60%;毒死蜱·噻虫嗪水分散粒剂则仅抑制CarE和GSTs活力,抑制率为71.16%和88.55%;3种复配剂明显影响菜青虫中肠肠壁细胞的层次结构以及肠壁细胞中的杯状细胞形状及结构.[结论]3种新型复配剂可能是通过抑制菜青虫体内关键酶活性和破坏中肠组织细胞形状和结构,进而影响其正常的消化和吸收功能,达到增效和杀虫的目的.%[Methods] In order to explicit their synergisms and mechanisms against Pieris rapae L., the synergized action against Pieris rapae L. Of three new mixtures of acephate and thiamethoxam emulsion in water, acephate and thiamethoxam water dispersible granule, chlorpyrifos and thiamethoxam water dispersible granule were determined by using spray application bioassays. The influences on the activities of carboxylesterase(CarE), glutathione S-transferase (GSTs) and acetylcholinesterase(AChE) were tested by biochemical method. Besides, the effects on midgut cells were studied by paraffin section method. [Results] All the three mixtures showed good insecticidal activity on P. Rapae L.. The co-toxicity coefficient (CTCs

  17. 甲氧虫酰肼对舞毒蛾幼虫解毒酶及其体内蛋白质表达的影响%Effect of Methoxyfenozide on Activities of Detoxifying Enzymes and Expression of Proteins in Lymantria dispar larvae

    Institute of Scientific and Technical Information of China (English)

    廖月枝; 严善春; 曹传旺; 刘丹

    2012-01-01

    In order to study the insecticidal activity of methoxyfenozide ( RH-2485 ) against the larvae of Lymantria dispar ,the pesticide bioactivity of the chemical against the different instar larvae of L. dispar and its effect on the activities of detoxifying enzymes of the insect were assayed using leaf film method, and the expression of proteins in different tissues of the larvae were detected with SDS-PAGE. The results showed that methoxyfenozide had a high toxicity against the larvae,especially 2nd instar and 3rd instar, and the toxicity was obviously different with different larva instars. The activities of detoxifying enzymes, such as carboxylesterase ( CarE ) , MFO O-demethylase ( MFOD ) and glutathione S-transfer (GST) , in the 2nd , 4th , 6th instar larvae were significantly induced or inhibited by the methoxyfenozide treatment. The impact of methoxyfenozide on these enzymes was significantly different at different treatment time. After the 4th instar larvae were fed with methoxyfenozide, the protein expression pattern in the hemolymph ,midgut and epidermis was different from that in the control. The effect of methoxyfenozide on proteins in the hemolymph and midgut was obvious in 12 h and 24 h, whereas the effect on protein expression in epidermis tissue was more significant in 48 h. These results indicated that methoxyfenozide as a non-steroidal ecdysone had a higher biological activity against L. dispar, and the major detoxifying enzymes in the insect body were significantly interfered, showing that methoxyfenozide had a high toxic effect against L. dispar. The specific proteins were produced in the hemolymph , midgut and epidermal tissue, which might interfere with the normal physiological metabolism of insects and epidermal formation.%为研究甲氧虫酰肼(RH-2485)对舞毒蛾幼虫的杀虫活性,采用叶片药膜法测定该药剂对舞毒蛾不同龄期幼虫的生物活性及对其体内解毒酶活性的影响,并通过SDS-PAGE对舞毒

  18. 菜蛾绒茧蜂和小菜蛾对杀虫剂的敏感性及酶学特性的比较研究%Susceptibility to insecticides and enzymetic characteristics in the parasitoid Apanteles plutellae Kurdj.(Hymenoptera:Braconidae) and its host Plutella xylostella (L.)( Lepidoptera:Yponomeutidae)

    Institute of Scientific and Technical Information of China (English)

    吴刚; 江树人

    2004-01-01

    The susceptibility to insecticides in the larval parasitoid Apanteles plutellae Kurdjumov (Hymenoptera: Braconidae)and its host Plutella xylostella (L.) (Lepidoptera: Yponomeutidae),collected in Fuzhou, China, were detected using residual film and leaf-dip bioassays, respectively. The results showed that organophosphates, carbamates, pyrethroids, avermectins and fipronil were highly toxic to A. plutellae, but chlorfluazuron and Bt were not. However, A. plutellae could survive from the conventional control doses of fipronil, fenvalerate, cypermethrin and acephate if the parasitoid was left in contact with the insecticides only for short time ( 1 h). In A. plutellae, there were obvious synergisms of piperonyl butoxide ( PB), triphenyl phosphate (TPP) and diethyl maleate (DEM) on methamidophos, carbofuran, fenvalerate, cypermethrin, avermectins and fipronil, but no synergisms on chlorfluazuron were found. The synergism of PB was the highest. Acetylcholinesterase (AChE)activity could not be inhibited by PB, TPP and DEM, but strong inhibition could be found in carboxylesterase (CarE) activity by PB and TPP, and in glutathione-S-transferase (GST) activity by DEM, in vivo. The apparent Michaelis-Menten constant ( Km ),the maximal velocity (Vmax) of AChE, and the activities of CarE and GST in A. plutellae were 0.22, 2.08, 4.60 and 0.45-fold as those in P. xylostella, respectively. The bimolecular rate constants (Ki ) of AChE to methamidophos, dichlorvos and carbofuran in A. plutellae were 14.7, 10.5 and 26.0-fold as those in P. xylostella, respectively. High inhibition of AChE was found in both species when being incubated with insecticides at high temperature, especially in A. plutellae. The results indicated that the high susceptibilities to organophosphates and carbametes in A. plutellae were related to its high sensitivity of AChE to the insecticides, and the oxidative metabolism might be more effective in tolerance to insecticides than non-oxidative metabolism in A

  19. Toxicity of cycloxaprid toAphis craccivora (Koch) and its effects on detoxification enzymes%环氧虫啶对苜蓿蚜的毒力及对其体内解毒酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    吴勇超; 须志平; 邵旭升; 程家高; 李忠

    2016-01-01

    tests revealed that the activities of detoxification enzymes, namely, glutathioneS-transferase (GSTs) and cytochrome P450 enzymes (7-ethoxycoumarin-O-deethylase) inA. craccivora, could be enhanced significantly (P< 0.05) by the treatment of cycloxaprid and imidacloprid (LC50 level). Although cycloxaprid could enhance the activities of the above two enzymes to (2.730 ± 0.012) and (0.239 ± 0.009) μmol/(mg pro.·min), respectively, it is still lower than that of imidacloprid. No significant change was observed for the activities of carboxylesterase (CarE). The above results indicated that GSTs and P450s might play key roles during the detoxification of cyclcxaprid inA. craccivora.

  20. 苍耳甾醇物质对菜青虫取食、血淋巴和中肠酶活性及中肠组织的影响%Effects of sterols from Xanthium sibiricum (Compositae) on feeding,enzyme activities in the hemolymph and midgut, and midgut tissues of Pieris rapae ( Lepidoptera: Pieridae) larvae

    Institute of Scientific and Technical Information of China (English)

    周琼; 熊正燕; 欧晓明

    2011-01-01

    甾醇是植物体内的重要次生物质,具有多种生物活性.为探明植物甾醇类物质对害虫的作用机理,采用叶碟饲喂法进行取食处理后研究了苍耳Xanthium sibiricum中分离纯化的甾醇类组分(甾醇A和甾醇B)对4龄菜青虫Pieris rapae的取食、酶活性以及中肠组织的影响.结果表明:苍耳甾醇类组分甾醇A和甾醇B能明显抑制菜青虫的取食,拒食中浓度AFC50分别为0.0229和0.0147 mg/mL;同时,显著降低菜青虫中肠蛋白酶、淀粉酶和羧酸酯酶活性,其中,甾醇B的作用效果较强,处理后24h和36 h,对蛋白酶活性抑制率分别为23.74%和58.59%,对中肠羧酸酯酶的活性抑制率分别为49.01%和83.03%;降低血淋巴蛋白质含量,诱导菜青虫血淋巴羧酸酯酶活性的提高;破坏昆虫中肠上皮组织,微杆模糊不清呈消融状,杯状细胞的杯腔基部微绒毛消失.这些结果说明苍耳甾醇类物质对菜青虫的取食抑制可能与对中肠消化酶活性的抑制以及对中肠上皮组织的破坏有关,植物甾醇组分的不同配比影响其对昆虫的作用效果.%Sterols are important secondary metabolites in plants with a variety of biological activities. In order to reveal the mechanism of sterols from plants against insect pests, the effects of sterol constituents ( sterol A and sterol B) extracted from Xanthium sibiricum ( Compositae) on feeding, enzyme activities in the hemolymph and midgut, and midgut tissues of 4th instar larvae of Pieris rapae treated by feeding on leaf-discs of crucifer Brassica oleracea were investigated. The results showed that the feeding of P. Rapae larvae was obviously deterred by sterol A and sterol B with the AFC50 of 0. 0229 mg/mL and 0. 0147 mg/mL, respectively, at 24 h after treatment. The activities of midgut amylase, protease and carboxylesterase (CarE) were significantly lower than those of the control within 36 h treatment, and sterol B showed stronger effects, which inhibited

  1. 细辛精油对2种农业害虫保护酶和解毒酶活性的影响%Effects of Asarum Essential Oils on Protection/Detoxification Enzyme Activities of Two Agricultural Pest Insects

    Institute of Scientific and Technical Information of China (English)

    姬兰柱; 王桂清; 刘艳; 王远遐; 张悦; 易雪梅

    2013-01-01

    To evaluate the insecticidal potential and mechanism of asarum essential oils ,the effects of three kinds of asarum essential oils extracted by different methods on protection/detoxification enzyme activities of Asian corn borer[Ostrinia furnacalis(Guenee)] and army worm[Mythimna seperata(Walker)] were studied .The three asarum essential oils were as follows :essential oil X1 was extracted by steam distillation method ,essential oil X3 was extracted by supercritical CO2 ex-traction method ,and essential oil X5 was a product obtained by refining X3 through a silica gel column chromatography .X1 was sprayed on the corn sprout ,according to a dose of 1 000 mg/L . X3 and X5 were separately added to the corn borer artificial feed ,according to a dose of 0 .5 mg/g . The experiment tested the response of Asian corn borer larvae fed on artificial diet containing X 3 or X5 ,and response of army worm larvae fed on tender corn shoots containing X 1 .In control group ,asarum essential oil was not added .The results showed that ,under the set experimental concentrations ,essential oils X1 and X3 inhibited the activities of three protective enzymes (su-peroxide dismutase ,SOD ;catalase ,CAT ;peroxidase ,POD) of the test insects ;X5 showed cer-tain inhibitory effect on POD activity of the test insects ,but exhibited an activation effect on SOD and CAT .Asarum essential oils showed some activation effect on carboxylesterase (CarE) ,acid phosphatase (ApE) and glutathione S-transferase (GST ) ,but the degree of activation effect re-duced over time ,meanwhile alkaline phosphatase(ALP) activity significantly increased early ,then significantly reduced ,w hich demonstrated that insects firstly increased their detoxification ability under excessive exogenous compound ,and then the detoxification capacity would decrease with the accumulation of toxic substances .The insecticidal effect of asarum essential oils may be achieved by inhibiting the insects’ protective enzymes .%为评价

  2. Final amended report on the safety assessment of Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben as used in cosmetic products.

    Science.gov (United States)

    2008-01-01

    Parabens is the name given to a group of p-hydroxybenzoic acid (PHBA) esters used in over 22,000 cosmetics as preservatives at concentrations up to 0.8% (mixtures of parabens) or up to 0.4% (single paraben). The group includes Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben. Industry estimates of the daily use of cosmetic products that may contain parabens were 17.76 g for adults and 378 mg for infants. Parabens in cosmetic formulations applied to skin penetrate the stratum corneum in inverse relation to the ester chain length. Carboxylesterases hydrolyze parabens in the skin. Parabens do not accumulate in the body. Serum concentrations of parabens, even after intravenous administration, quickly decline and remain low. Acute toxicity studies in animals indicate that parabens are not significantly toxic by various routes of administration. Subchronic and chronic oral studies indicate that parabens are practically nontoxic. Numerous genotoxicity studies, including Ames testing, dominant lethal assay, host-mediated assay, and cytogenic assays, indicate that the Parabens are generally nonmutagenic, although Ethylparaben and Methylparaben did increase chromosomal aberrations in a Chinese Hamster ovary cell assay. Ethylparaben, Propylparaben, and Butylparaben in the diet produced cell proliferation in the forestomach of rats, with the activity directly related to chain length of the alkyl chain, but Isobutylparaben and Butylparaben were noncarcinogenic in a mouse chronic feeding study. Methylparaben was noncarcinogenic when injected subcutaneously in mice or rats, or when administered intravaginally in rats, and was not cocarcinogenic when injected subcutaneously in mice. Propylparaben was noncarcinogenic in a study of transplacental carcinogenesis. Methylparaben was nonteratogenic in rabbits, rats, mice, and hamsters, and Ethylparaben was nonteratogenic in rats. Parabens, even at levels that produce maternal