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Sample records for acetylcholine esterase inhibitors

  1. Organophosphate acetylcholine esterase inhibitor poisoning from a home-made shampoo.

    PubMed

    Sadaka, Yair; Broides, Arnon; Tzion, Raffi Lev; Lifshitz, Matitiahu

    2011-07-01

    Organophosphate acetylcholine esterase inhibitor poisoning is a major health problem in children. We report an unusual cause of organophosphate acetylcholine esterase inhibitor poisoning. Two children were admitted to the pediatric intensive care unit due to organophosphate acetylcholine esterase inhibitor poisoning after exposure from a home-made shampoo that was used for the treatment of head lice. Owing to no obvious source of poisoning, the diagnosis of organophosphate acetylcholine esterase inhibitor poisoning in one of these patients was delayed. Both patients had an uneventful recovery. Organophosphate acetylcholine esterase inhibitor poisoning from home-made shampoo is possible. In cases where the mode of poisoning is unclear, direct questioning about the use of home-made shampoo is warranted, in these cases the skin and particularly the scalp should be rinsed thoroughly as soon as possible.

  2. Acetylcholine esterase inhibitors in effluents from oil production platforms in the North Sea.

    PubMed

    Holth, T F; Tollefsen, K E

    2012-05-15

    Inhibition of acetylcholine esterase (AChE) activity is a biomarker for the exposure to neurotoxic compounds such as organophosphates and is intimately associated with the toxicity of several pesticides. In the present study, the AChE inhibiting potential of organic extracts of production water (produced water) from oil and gas production platforms in the Norwegian sector of the North Sea was determined in an in vitro bioassay based on commercially available purified AChE from the electric organ of Electrophorus electricus (L.). The results from the studies show that produced water contains a combination of AChE inhibiting compounds and compounds stimulating AChE enzymatic activity. The AChE inhibition was predominantly caused by unidentified aromatic compounds in the oil/particulate fraction of produced water, whereas polar compounds in both the water soluble and oil/particulate fraction of produced water caused an apparent stimulation of AChE activity. Substrate saturation studies with fixed concentrations of produced water extracts confirmed that the inhibition occurred in a non-destructive and competitive manner. The concentrations of AChE inhibitors (7.9-453 ng paraoxon-equivalents L⁻¹, 2.2-178 μg dichlorvos-equivalents L⁻¹) were in many cases found to be several orders of magnitude higher than background levels. The findings demonstrate that produced water contains potentially neurotoxic compounds and suggest that further laboratory studies with fish or field studies in the vicinity of oil production facilities are highly warranted.

  3. In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat.

    PubMed

    Killi, Uday K; Wsol, Vladimir; Soukup, Ondrej; Kuca, Kamil; Winder, Michael; Tobin, Gunnar

    2014-02-01

    Obidoxime, a weak acetylcholine-esterase (AChE) inhibitor, exerts muscarinic receptor antagonism with a significant muscarinic M2 receptor selective profile. The current examinations aimed to determine the functional significance of muscarinic M2 receptors in the state of AChE inhibition, elucidating muscarinic M2 and M3 receptor interaction. In the in vitro examinations, methacholine evoked concentration-dependent bladder contractile and atrial frequency inhibitory responses. Although atropine abolished both, methoctramine (1 μmol/L) only affected the cholinergic response in the atrial preparations. However, in the presence of methoctramine, physostigmine, an AChE inhibitor, increased the basal tension of the bladder strip preparations (+68%), as well as the contractile responses to low concentrations of methacholine (< 5 μmol/L; +90-290%). In contrast to physostigmine, obidoxime alone raised the basal tension (+58%) and the responses to low concentrations of methacholine (< 5 μmol/L; +80-450%). Physostigmine concentration-dependently increased methacholine-evoked responses, similarly to obidoxime at low concentrations. However, at large concentrations (> 5 μmol/L), obidoxime, because of its unselective muscarinic receptor antagonism, inhibited the methacholine bladder responses. In conclusion, the current results show that muscarinic M2 receptors inhibit muscarinic M3 receptor-evoked contractile responses to low concentrations of acetylcholine in the synaptic cleft. The muscarinic M2 and M3 receptor crosstalk could be a counteracting mechanism in the treatment of AChE inhibition when using reactivators, such as obidoxime.

  4. Atomic insight into designed carbamate-based derivatives as acetylcholine esterase (AChE) inhibitors: a computational study by multiple molecular docking and molecular dynamics simulation.

    PubMed

    Mohammadi, Tecush; Ghayeb, Yousef

    2017-01-11

    Over 100 variants have been designed and studied, using multiple docking methods such as Autodock Vina, ArgusLab, Molegro Virtual Docker, and Hex-Cuda, to study the effect of alteration in the structure of carbamate-based acetylcholyne esterase (AChE) inhibitors. Sixteen selected systems were then subjected to 14 ns molecular dynamics (MD) simulations. Results from all the docking methods are in agreement. Variants that involved biphenyl substituents possess the most negative binding energies in the -37.64 to -39.31 kJ mol(-1) range due to their π-π interactions with AChE aromatic residues. The root mean square deviation values showed that all of these components achieved equilibration after 6 ns. Gyration radius (Rg) and solvent accessibility surface area were calculated to further investigate the AChE conformational changes in the presence of these components. MD simulation results suggested that these components might interact with AChE, possibly with no major changes in AChE secondary and tertiary structures.

  5. [Influence of UV-light on erythrocyte membrane structure and catalytic behaviour of membrane acetylcholine esterase].

    PubMed

    Volotovskiĭ, I D; Sheĭko, L M; Konev, S V

    1976-01-01

    UV-light is shown to induce the structural transitions in the erythrocyte membrane described by S-shape curves in plots of the structural response versus the irradiation dose. In contrast to the free acetylcholine esterase (AChE) UV-light acts on the membrane enzyme as a mixed inhibitor (simultaneous change in Vmax and Km). The modification of the environment structure of residual enzyme is suggested to be the main reason of this phenomenon. The effect is under the control of membrane integrity and disappears after its desintegration. Membrane AChE treated ultrasonically both prior to and after irradiation is inactivated without a Km change. The data obtained show the influence of erythrocyte membrane structure on the catalytic behaviour of membrane-bound AChE.

  6. Esterase detoxification of acetylcholinesterase inhibitors by ...

    EPA Pesticide Factsheets

    Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON) are considered factors underlying age-related sensitivity differences. We used an in vitro system to measure detoxification of AChE-inhibiting pesticides mediated via these esterases. Recombinant human AChE was used as a bioassay of inhibitor concentration following incubation with detoxifying tissue: liver plus Ca+2 (to stimulate PONs, measuring activity of both esterases) or EGTA (to inhibit PONs, thereby measuring CaE activity). Inhibitory concentrations of aldicarb, chlorpyrifos oxon, malaoxon, methamidophos, oxamyl, paraoxon, and methyl paraoxon were incubated with liver from adult male rat or one of 20 commercially provided human (11-83 years of age) liver samples. Detoxification was the difference in inhibition produced by the pesticide alone or in combination with liver plus Ca+2 or EGTA. Generally, rat liver produced more detoxification than did the human samples. There were large detoxification differences, which were not correlated with age or sex, across human samples for some pesticides (especially malaoxon, chlorpyrifos oxon) but not for others (e.g., aldicarb, methamidophos). Chlorpyrifos oxon was detoxified only in the presence of Ca+2 in both rat and human livers. Detoxification of pa

  7. Donepezil, an acetylcholine esterase inhibitor, and ABT-239, a histamine H3 receptor antagonist/inverse agonist, require the integrity of brain histamine system to exert biochemical and procognitive effects in the mouse.

    PubMed

    Provensi, Gustavo; Costa, Alessia; Passani, M Beatrice; Blandina, Patrizio

    2016-10-01

    Histaminergic H3 receptors (H3R) antagonists enhance cognition in preclinical models and modulate neurotransmission, in particular acetylcholine (ACh) release in the cortex and hippocampus, two brain areas involved in memory processing. The cognitive deficits seen in aging and Alzheimer's disease have been associated with brain cholinergic deficits. Donepezil is one of the acetylcholinesterase (AChE) inhibitor approved for use across the full spectrum of these cognitive disorders. We addressed the question if H3R antagonists and donepezil require an intact histamine neuronal system to exert their procognitive effects. The effect of the H3R antagonist ABT-239 and donepezil were evaluated in the object recognition test (ORT), and on the level of glycogen synthase kinase 3 beta (GSK-3β) phosphorylation in normal and histamine-depleted mice. Systemic administration of ABT-239 or donepezil ameliorated the cognitive performance in the ORT. However, these compounds were ineffective in either genetically (histidine decarboxylase knock-out, HDC-KO) or pharmacologically, by means of intracerebroventricular (i.c.v.) injections of the HDC irreversible inhibitor a-fluoromethylhistidine (a-FMHis), histamine-deficient mice. Western blot analysis revealed that ABT-239 or donepezil systemic treatments increased GSK-3β phosphorylation in cortical and hippocampal homogenates of normal, but not of histamine-depleted mice. Furthermore, administration of the PI3K inhibitor LY294002 that blocks GSK-3β phosphorylation, prevented the procognitive effects of both drugs in normal mice. Our results indicate that both donepezil and ABT-239 require the integrity of the brain histaminergic system to exert their procognitive effects and strongly suggest that impairments of PI3K/AKT/GSK-3β intracellular pathway activation is responsible for the inefficacy of both drugs in histamine-deficient animals.

  8. Effects on operant learning and brain acetylcholine esterase activity in rats following chronic inorganic arsenic intake.

    PubMed

    Nagaraja, T N; Desiraju, T

    1994-05-01

    1. Very young and adult Wistar rats were given As5+, 5 mg arsenic kg-1 body weight day-1 (sodium arsenate). 2. Operant learning was tested in a Skinner box at the end of exposure and, in the case of developing animals, also after a recovery period. 3. Acetylcholine esterase (AChE) activity was estimated in discrete brain regions of these animals. 4. The animals exposed to arsenic took longer to acquire the learned behaviour and to extinguish the operant. AChE activity was inhibited in some regions of the brain.

  9. Automated high-throughput in vitro screening of the acetylcholine esterase inhibiting potential of environmental samples, mixtures and single compounds.

    PubMed

    Froment, Jean; Thomas, Kevin V; Tollefsen, Knut Erik

    2016-08-01

    A high-throughput and automated assay for testing the presence of acetylcholine esterase (AChE) inhibiting compounds was developed, validated and applied to screen different types of environmental samples. Automation involved using the assay in 96-well plates and adapting it for the use with an automated workstation. Validation was performed by comparing the results of the automated assay with that of a previously validated and standardised assay for two known AChE inhibitors (paraoxon and dichlorvos). The results show that the assay provides similar concentration-response curves (CRCs) when run according to the manual and automated protocol. Automation of the assay resulted in a reduction in assay run time as well as in intra- and inter-assay variations. High-quality CRCs were obtained for both of the model AChE inhibitors (dichlorvos IC50=120µM and paraoxon IC50=0.56µM) when tested alone. The effect of co-exposure of an equipotent binary mixture of the two chemicals were consistent with predictions of additivity and best described by the concentration addition model for combined toxicity. Extracts of different environmental samples (landfill leachate, wastewater treatment plant effluent, and road tunnel construction run-off) were then screened for AChE inhibiting activity using the automated bioassay, with only landfill leachate shown to contain potential AChE inhibitors. Potential uses and limitations of the assay were discussed based on the present results.

  10. Transition State Analog Inhibitors for Esterases.

    DTIC Science & Technology

    1983-06-02

    advanced about 1970, it has led to the synthesis of powerful reversible Inhibitors for a number of enzymes. More recently, transition state analog theory...to inactivate It. Organophosphate anticholinesterases are a classic example; many of the more recent examples allow the enzyme to generate a strong...could be utilized to explore such mechanistic questions. A second more practical goal was to prepare anticholinesterases of novel structure and

  11. 3 Benzyl-6-chloropyrone: a suicide inhibitor of cholesterol esterase

    SciTech Connect

    Saint, C.; Gallo, I.; Kantorow, M.; Bailey, J.M.

    1986-05-01

    Cholesterol, absorbed from the intestine, appears in lymph as the ester. Cholesterol esterase is essential for this process, since depletion of the enzyme blocks and repletion restores, absorption. Selective inhibitors of cholesterol esterase may thus prove useful in reducing cholesterol uptake. A series of potential suicide substrates were synthesized which, following cleavage by the enzyme, would attack the putative nucleophile in the active site. One of these, 3-benzyl-6-chloropyrone (3BCP), inhibited both synthesis and hydrolysis of /sup 14/C-cholesteryl oleate with an I/sub 50/ of approximately 150 ..mu..M. The inactivation was time-dependent and characteristic of a suicide mechanism. The ..cap alpha.. pyrone structure (lactone analog) is cleaved by a serine-hydroxyl in the active site. This generates an enoyl chloride which inactivates the imidazole believed to play a part in the catalytic function of the enzyme. Inhibition by 3BCP is selective for cholesterol esterase. The activity of pancreatic lipase as not affected by concentrations up to 1 mM.

  12. In vivo study of acetylcholine esterase in basal forebrain, amygdala, and cortex in mild to moderate Alzheimer disease.

    PubMed

    Herholz, K; Weisenbach, S; Zündorf, G; Lenz, O; Schröder, H; Bauer, B; Kalbe, E; Heiss, W-D

    2004-01-01

    It is currently unclear whether impairment of the cholinergic system is present in Alzheimer disease (AD) already at an early stage and to what extent it depends on degeneration of the nucleus basalis of Meynert (nbM). We examined acetylcholine esterase activity in vivo in the nbM, the amygdala, and cerebral neocortex. Measurements were performed in normal controls and in patients with mild to moderate AD with positron emission tomography (PET) and C-11-labeled N-methyl-4-piperidyl-acetate (MP4A) which is a specific substrate of AChE. AChE activity was reduced significantly in amygdala and cerebral cortex. In contrast, AChE activity and glucose metabolism appeared preserved or even increased in the nbM. The results support the concept that neocortical and amygdaloid functional changes of the cholinergic system are an early and leading event in AD, rather than the consequence of neurodegeneration of basal nuclei.

  13. Acetylcholine esterase activity and behavioral response in hypoxia induced neonatal rats: effect of glucose, oxygen and epinephrine supplementation.

    PubMed

    Chathu, Finla; Krishnakumar, Amee; Paulose, Cheramadathikudyil S

    2008-10-01

    Brain damage due to an episode of hypoxia remains a major problem in infants causing deficit in motor and sensory function. Hypoxia leads to neuronal functional failure, cerebral palsy and neuro-developmental delay with characteristic biochemical and molecular alterations resulting in permanent or transitory neurological sequelae or even death. During neonatal hypoxia, traditional resuscitation practices include the routine administration of 100% oxygen, epinephrine and glucose. In the present study, we assessed the changes in the cholinergic system by measuring the acetylcholinesterase (AChE) activity and the behavioral responses shown by hypoxia induced neonatal rats and hypoxic rats supplemented with glucose, oxygen and epinephrine using elevated plus-maze and open-field test. The acetylcholine esterase enzyme activity showed a significant decrease in cerebral cortex, whereas it increased significantly in the muscle of experimental rats when compared to control. Hypoxic rats supplemented with glucose, glucose and oxygen showed a reversal to the control status. Behavioral studies were carried out in experimental rats with elevated plus-maze test and open-field test. Hypolocomotion and anxiogenic behavioral responses were observed in all experimental rats when compared to control, hypoxic rats supplemented with glucose, glucose and oxygen. Thus, our results suggest that brain damage due to hypoxia, oxygen and epinephrine supplementation in the neonatal rats cause acetylcholine-neuromuscular-defect leading to hypolocomotion and anxiogenic behavioral response. Glucose and glucose with oxygen supplementation to hypoxic neonates protect the brain damage for a better functional status in the later life.

  14. Effects of three reputed carboxylesterase inhibitors upon rat serum esterase activity.

    PubMed

    Chambers, J P; Hartgraves, S L; Murphy, M R; Wayner, M J; Kumar, N; Valdes, J J

    1991-01-01

    Rats have very high endogenous levels of serum carboxylesterase (CAE) compared to primates. This difference accounts for the lower sensitivity of rats to toxic organophosphates, which interact with CAE instead of the more critical acetylcholinesterase. Pretreatment of rats with CAE inhibitors potentiates the effects of organophosphates. In this study, the effects of three putative CAE inhibitors, 2-(o-Cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP), bis-p-nitrophenyl-phosphate (BNPP), and tetraisopropyl pyrophosphoramide (Iso-OMPA), on the hydrolysis of several commercially available substrates were determined. Respective kinetic constants Km and Vmax were derived and effects of inhibitors compared using saturating amounts of substrate. Data presented here indicate significant differences in substrate affinity (Km), reactivity (Vmax), as well as effects of inhibitors. CBDP inhibits hydrolysis of specific naphthyl and paranitrophenyl esters at relatively low concentrations (1-10 microM). In contrast, significantly higher concentrations (mM) of BNPP and Iso-OMPA were required for inhibition of serum esterase activity. Of the inhibitors tested, Iso-OMPA in general exhibited the smallest inhibitory effect on ester hydrolysis. Although inhibition of hydrolysis of specific paranitrophenyl and naphthyl esters occurred in the presence of similar amounts of CBDP, the degree of inhibition differed significantly (50-75% vs. greater than 90%, respectively). These data suggest that there exists in rat serum, a pool of naphthyl ester esterase activity that is very sensitive ex vivo (greater than 90% inhibition) to CBDP and may be very useful in validating a rodent model for soman toxicity.

  15. Resistance mechanisms to chlorpyrifos and F392W mutation frequencies in the acetylcholine esterase ace1 allele of field populations of the tobacco whitefly, Bemisia tabaci in China.

    PubMed

    Zhang, Ning-ning; Liu, Cai-feng; Yang, Fang; Dong, Shuang-lin; Han, Zhao-jun

    2012-01-01

    The tobacco whitefly B-biotype Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a worldwide pest of many crops. In China, chlorpyrifos has been used to control this insect for many years and is still being used despite the fact that some resistance has been reported. To combat resistance and maintain good control efficiency of chlorpyrifos, it is essential to understand resistance mechanisms. A chlorpyrifos resistant tobacco whitefly strain (NJ-R) and a susceptible strain (NJ-S) were derived from a field-collected population in Nanjing, China, and the resistance mechanisms were investigated. More than 30-fold resistance was achieved after selected by chlorpyrifos for 13 generations in the laboratory. However, the resistance dropped significantly to about 18-fold in only 4 generations without selection pressure. Biochemical assays indicated that increased esterase activity was responsible for this resistance, while acetylcholine esterase, glutathione S-transferase, and microsomal-O-demethylase played little or no role. F392W mutations in acel were prevalent in NJ-S and NJ-R strains and 6 field-collected populations of both B and Q-biotype from locations that cover a wide geographical area of China. These findings provide important information about tobacco whitefly chlorpyrifos resistance mechanisms and guidance to combat resistance and optimize use patterns of chlorpyrifos and other organophosphate and carbamate insecticides.

  16. Resistance Mechanisms to Chlorpyrifos and F392W Mutation Frequencies in the Acetylcholine Esterase Ace1 Allele of Field Populations of the Tobacco Whitefly, Bemisia tabaci in China

    PubMed Central

    Zhang, Ning-ning; Liu, Cai-feng; Yang, Fang; Dong, Shuang-lin; Han, Zhao-jun

    2012-01-01

    The tobacco whitefly B-biotype Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a worldwide pest of many crops. In China, chlorpyrifos has been used to control this insect for many years and is still being used despite the fact that some resistance has been reported. To combat resistance and maintain good control efficiency of chlorpyrifos, it is essential to understand resistance mechanisms. A chlorpyrifos resistant tobacco whitefly strain (NJ-R) and a susceptible strain (NJ-S) were derived from a field-collected population in Nanjing, China, and the resistance mechanisms were investigated. More than 30-fold resistance was achieved after selected by chlorpyrifos for 13 generations in the laboratory. However, the resistance dropped significantly to about 18-fold in only 4 generations without selection pressure. Biochemical assays indicated that increased esterase activity was responsible for this resistance, while acetylcholine esterase, glutathione S-transferase, and microsomal-O-demethylase played little or no role. F392W mutations in acel were prevalent in NJ-S and NJ-R strains and 6 field-collected populations of both B and Q-biotype from locations that cover a wide geographical area of China. These findings provide important information about tobacco whitefly chlorpyrifos resistance mechanisms and guidance to combat resistance and optimize use patterns of chlorpyrifos and other organophosphate and carbamate insecticides. PMID:22954331

  17. Angiotensin-converting enzyme inhibitors-induced angioedema treated by C1 esterase inhibitor concentrate (Berinert®): about one case and review of the therapeutic arsenal.

    PubMed

    Lipski, Samuel Michael; Casimir, Georges; Vanlommel, Martine; Jeanmaire, Mathieu; Dolhen, Pierre

    2015-02-01

    C1 esterase inhibitor (Berinert®) is generally used to treat severe attack of hereditary angioedema. We describe here the case of a patient who presented with a severe angioedema induced by angiotensin-converting enzyme inhibitors (ACEIs) endangering her life. It could be successfully treated with that medicine.

  18. Insecticidal and acetylcholine esterase inhibition activity of Apiaceae plant essential oils and their constituents against adults of German cockroach (Blattella germanica).

    PubMed

    Yeom, Hwa-Jeong; Kang, Jae Soon; Kim, Gil-Hah; Park, Il-Kwon

    2012-07-25

    We evaluated the insecticidal and acetylcholine esterase (AChE) inhibition activity of 11 Apiaceae plant essential oils and their constituents in adult male and female Blattella germanica. Of the 11 Apiaceae plant essential oils tested, dill (Anethum graveolens), carvi (Carum carvi), and cumin (Cuminum cyminum) demonstrated >90% fumigant toxicity against adult male German cockroaches at a concentration of 5 mg/filter paper. In a contact toxicity test, dill (Anethum graveolens), carvi (Carum carvi), cumin (Cuminum cyminum), and ajowan (Trachyspermum ammi) produced strong insecticidal activity against adult male and female German cockroaches. Among the test compounds, (S)-(+)-carvone, 1,8-cineole, trans-dihydrocarvone, cuminaldehyde, trans-anethole, p-cymene, and γ-terpinene demonstrated strong fumigant toxicity against adult male and female B. germanica. In a contact toxicity test, carveol, cuminaldehyde, (S)-(+)-carvone, trans-anethole, thymol, and p-cymene showed strong contact toxicity against adult male and female B. germanica. IC(50) values of α-pinene, carvacrol, and dihydrocarvone against female AChE were 0.28, 0.17, and 0.78 mg/mL, respectively. The toxicity of the blends of constituents identified in 4 active oils indicated that carvone, cuminaldehyde, and thymol were major contributors to the fumigant activity or contact toxicity of the artificial blend.

  19. Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

    PubMed Central

    Marzin, Stephan; Hanemann, Anja; Sharma, Shailendra; Hensel, Götz; Kumlehn, Jochen; Schweizer, Günther; Röder, Marion S.

    2016-01-01

    A family of putative PECTIN ESTERASE INHIBITOR (PEI) genes, which were detected in the genomic region co-segregating with the resistance gene Rrs2 against scald caused by Rhynchosporium commune in barley, were characterized and tested for their possible involvement in mediating resistance to the pathogen by complementation and overexpression analysis. The sequences of the respective genes were derived from two BAC contigs originating from the susceptible cultivar ‘Morex’. For the genes HvPEI2, HvPEI3, HvPEI4 and HvPEI6, specific haplotypes for 18 resistant and 23 susceptible cultivars were detected after PCR-amplification and haplotype-specific CAPS-markers were developed. None of the tested candidate genes HvPEI2, HvPEI3 and HvPEI4 alone conferred a high resistance level in transgenic over-expression plants, though an improvement of the resistance level was observed especially with OE-lines for gene HvPEI4. These results do not confirm but also do not exclude an involvement of the PEI gene family in the response to the pathogen. A candidate for the resistance gene Rrs2 could not be identified yet. It is possible that Rrs2 is a PEI gene or another type of gene which has not been detected in the susceptible cultivar ‘Morex’ or the full resistance reaction requires the presence of several PEI genes. PMID:26937960

  20. Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

    PubMed

    Marzin, Stephan; Hanemann, Anja; Sharma, Shailendra; Hensel, Götz; Kumlehn, Jochen; Schweizer, Günther; Röder, Marion S

    2016-01-01

    A family of putative PECTIN ESTERASE INHIBITOR (PEI) genes, which were detected in the genomic region co-segregating with the resistance gene Rrs2 against scald caused by Rhynchosporium commune in barley, were characterized and tested for their possible involvement in mediating resistance to the pathogen by complementation and overexpression analysis. The sequences of the respective genes were derived from two BAC contigs originating from the susceptible cultivar 'Morex'. For the genes HvPEI2, HvPEI3, HvPEI4 and HvPEI6, specific haplotypes for 18 resistant and 23 susceptible cultivars were detected after PCR-amplification and haplotype-specific CAPS-markers were developed. None of the tested candidate genes HvPEI2, HvPEI3 and HvPEI4 alone conferred a high resistance level in transgenic over-expression plants, though an improvement of the resistance level was observed especially with OE-lines for gene HvPEI4. These results do not confirm but also do not exclude an involvement of the PEI gene family in the response to the pathogen. A candidate for the resistance gene Rrs2 could not be identified yet. It is possible that Rrs2 is a PEI gene or another type of gene which has not been detected in the susceptible cultivar 'Morex' or the full resistance reaction requires the presence of several PEI genes.

  1. Nebulized C1-Esterase Inhibitor does not Reduce Pulmonary Complement Activation in Rats with Severe Streptococcus Pneumoniae Pneumonia.

    PubMed

    de Beer, Friso; Lagrand, Wim; Glas, Gerie J; Beurskens, Charlotte J P; van Mierlo, Gerard; Wouters, Diana; Zeerleder, Sacha; Roelofs, Joris J T H; Juffermans, Nicole P; Horn, Janneke; Schultz, Marcus J

    2016-12-01

    Complement activation plays an important role in the pathogenesis of pneumonia. We hypothesized that inhibition of the complement system in the lungs by repeated treatment with nebulized plasma-derived human C1-esterase inhibitor reduces pulmonary complement activation and subsequently attenuates lung injury and lung inflammation. This was investigated in a rat model of severe Streptococcus pneumoniae pneumonia. Rats were intra-tracheally challenged with S. pneumoniae to induce pneumonia. Nebulized C1-esterase inhibitor or saline (control animals) was repeatedly administered to rats, 30 min before induction of pneumonia and every 6 h thereafter. Rats were sacrificed 20 or 40 h after inoculation with bacteria. Brochoalveolar lavage fluid and lung tissue were obtained for measuring levels of complement activation (C4b/c), lung injury and inflammation. Induction of pneumonia was associated with pulmonary complement activation (C4b/c at 20 h 1.24 % [0.56-2.59] and at 40 h 2.08 % [0.98-5.12], compared to 0.50 % [0.07-0.59] and 0.03 % [0.03-0.03] in the healthy control animals). The functional fraction of C1-INH was detectable in BALF, but no effect was found on pulmonary complement activation (C4b/c at 20 h 0.73 % [0.16-1.93] and at 40 h 2.38 % [0.54-4.19]). Twenty hours after inoculation, nebulized C1-esterase inhibitor treatment reduced total histology score, but this effect was no longer seen at 40 h. Nebulized C1-esterase inhibitor did not affect other markers of lung injury or lung inflammation. In this negative experimental animal study, severe S. pneumoniae pneumonia in rats is associated with pulmonary complement activation. Repeated treatment with nebulized C1-esterase inhibitor, although successfully delivered to the lungs, does not affect pulmonary complement activation, lung inflammation or lung injury.

  2. Interaction of (benzylidene-hydrazono)-1,4-dihydropyridines with beta-amyloid, acetylcholine, and butyrylcholine esterases.

    PubMed

    Alptüzün, Vildan; Prinz, Michaela; Hörr, Verena; Scheiber, Josef; Radacki, Krzysztof; Fallarero, Adyary; Vuorela, Pia; Engels, Bernd; Braunschweig, Holger; Erciyas, Ercin; Holzgrabe, Ulrike

    2010-03-01

    Approved drugs for the treatment of Alzheimer's disease belong to the group of inhibitors of the acetylcholinesterase (AChE) and NMDA receptor inhibitors. However none of the drugs is able to combat or reverse the progression of the disease. Thus, the recently reported promising multitarget-directed molecule approach was applied here. Using the lead compound DUO3, which was found to be a potent inhibitor of the AChE and butyrylcholinesterase (BuChE) as well as an inhibitor of the formation of the amyloid (Abeta) plaque, new non-permanently positively charged derivatives were synthesized and biologically characterized. In contrast to DUO3 the new bisphenyl-substituted pyridinylidene hydrazones 5 are appropriate to cross the blood-brain barrier due to their pK(a) values and lipophilicity, and to inhibit both the AChE and BuChE. More important some of the pyridinylidene hydrazones inhibit the Abeta fibril formation completely and destruct the already formed fibrils significantly.

  3. Esterase detoxification of acetylcholinesterase inhibitors by human or rat liver in vitro

    EPA Science Inventory

    Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON) are considered...

  4. Protease inhibitor homologues from mamba venoms: facilitation of acetylcholine release and interactions with prejunctional blocking toxins.

    PubMed

    Harvey, A L; Karlsson, E

    1982-09-01

    1 Five polypeptides, which were isolated from elapid snake venoms and which are structurally related to protease inhibitors, were tested for action on isolated biventer cervicis nerve-muscle preparations of the chick. 2 Dendrotoxin from the Eastern green mamba (Dendroaspis angusticeps) and toxins K and I from the black mamba (Dendroaspis polylepis polylepis) increased to indirect stimulation without affecting responses to exogenous acetylcholine, carbachol of KCl. 3 The two other protease inhibitor homologues, HHV-II from Ringhals cobra (Hemachatus haemachatus) and NNV-II from Cape cobra (Naja nivea) did not increase responses to nerve stimulation. Trypsin inhibitor from bovine pancreas also had no facilitatory effects on neuromuscular transmission. 4 The facilitatory toxins from mamba venoms interacted with the prejunctional blocking toxins, beta-bungarotoxin, crotoxin and notexin, but not with taipoxin. The blocking effects of beta-bungarotoxin were reduced by pretreatment with the mamba toxins, whereas the blocking actions of crotoxin and notexin were enhanced. 5 The results indicate that protease inhibitor homologues from mamba venoms form a new class of neurotoxin, which acts to increase the release of acetylcholine in response to motor nerve stimulation. 6 From the interaction studies it is concluded that the facilitatory toxins bind to motor nerve terminals at sites related to those occupied by the prejunctional blocking toxins. However, differences in interactions with individual toxins suggest that there must be several related binding sites on the nerve terminals.

  5. Esterase detoxification of acetylcholinesterase inhibitors using human liver samples in vitro

    EPA Science Inventory

    Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are consider...

  6. Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

    PubMed

    Rocchi, V; Janni, M; Bellincampi, D; Giardina, T; D'Ovidio, R

    2012-03-01

    Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat.

  7. The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

    PubMed

    Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

    2011-09-01

    Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

  8. Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase.

    PubMed

    Swartz, Kenneth; Zhang, Yiguan; Valeriote, Frederick; Chen, Ben; Shaw, Jiajiu

    2013-12-01

    UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25°C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis-Menten kinetics showed that the Km values of UTL-5g were 2.07mM with PLE and 0.37mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA.

  9. Self-administered C1 esterase inhibitor concentrates for the management of hereditary angioedema: usability and patient acceptance

    PubMed Central

    Li, Huamin Henry

    2016-01-01

    Hereditary angioedema (HAE) is a rare genetic disease characterized by episodic subcutaneous or submucosal swelling. The primary cause for the most common form of HAE is a deficiency in functional C1 esterase inhibitor (C1-INH). The swelling caused by HAE can be painful, disfiguring, and life-threatening. It reduces daily function and compromises the quality of life of affected individuals and their caregivers. Among different treatment strategies, replacement with C1-INH concentrates is employed for on-demand treatment of acute attacks and long-term prophylaxis. Three human plasma-derived C1-INH preparations are approved for HAE treatment in the US, the European Union, or both regions: Cinryze®, Berinert®, and Cetor®; however, only Cinryze is approved for long-term prophylaxis. Postmarketing studies have shown that home therapy (self-administered or administered by a caregiver) is a convenient and safe option preferred by many HAE patients. In this review, we summarize the role of self-administered plasma-derived C1-INH concentrate therapy with Cinryze at home in the prophylaxis of HAE. PMID:27660422

  10. Tobacco nitrosamine N-nitrosonornicotine as inhibitor of neuronal nicotinic acetylcholine receptors.

    PubMed

    Nunes-Alves, Ariane; Nery, Arthur A; Ulrich, Henning

    2013-01-01

    Nitrosamines are well known for their carcinogenic potential. Recently, it was found that some of them may also interact with human nicotinic acetylcholine receptor (nAChR) subtypes. This work studied the effects of N-nitrosonornicotine (NNN) on recombinant rat α3β4 nAChR in HEK cells as well as on nAChR endogenously expressed in PC12 pheochromocytoma cells and in BC3H1 muscle-type cells. Whole-cell recording in combination with the cell-flow technique for agonist and inhibitor application in the millisecond time region revealed that NNN inhibits the activity of neuronal nAChR expressed in HEK or PC12, whereas weak inhibitory effects on muscle-type nAChR were observed at NNN concentrations up to 3 mM. Pharmacological actions of NNN and the inhibition mechanism were studied in detail using recombinant α3β4 nAChR expressed in HEK cells as a model. NNN-induced inhibition of nicotine-evoked α3β4 nAChR activity was dose-dependent with an inhibitory constant (IC(50)) of 0.92 ± 0.05 mM. Analysis based on mathematical models indicated a noncompetitive inhibition mechanism of the rat α3β4 nAChR by NNN. NNN's mechanism of action involves acceleration of conversion of the receptor from active to desensitized forms. In summary, this work shows that NNN inhibits rat α3β4 nAChR in a noncompetitive way and interacts weakly with muscular nAChR.

  11. Increased acetylcholine esterase activity produced by the administration of an aqueous extract of the seed kernel of Thevetia peruviana and its role on acute and subchronic intoxication in mice

    PubMed Central

    Marroquín-Segura, Rubén; Calvillo-Esparza, Ricardo; Mora-Guevara, José Luis Alfredo; Tovalín-Ahumada, José Horacio; Aguilar-Contreras, Abigail; Hernández-Abad, Vicente Jesús

    2014-01-01

    Background: The real mechanism for Thevetia peruviana poisoning remains unclear. Cholinergic activity is important for cardiac function regulation, however, the effect of T. peruviana on cholinergic activity is not well-known. Objective: To study the effect of the acute administration of an aqueous extract of the seed kernel of T. peruviana on the acetylcholine esterase (AChE) activity in CD1 mice as well its implications in the sub-chronic toxicity of the extract. Materials and Methods: A dose of 100 mg/kg of the extract was administered to CD1 mice and after 7 days, serum was obtained for ceruloplasmin (CP) quantitation and liver function tests. Another group of mice received a 50 mg/kg dose of the extract 3 times within 1 h time interval and AChE activity was determined for those animals. Heart tissue histological preparation was obtained from a group of mice that received a daily 50 mg/kg dose of the extract by a 30-days period. Results: CP levels for the treated group were higher than those for the control group (Student's t-test, P ≤ 0.001). AChE activity in the treated group was significantly higher than the control group (Tukey test, control vs. T. peruviana, P ≤ 0.001). Heart tissue histological preparations showed leukocyte infiltrates and necrotic areas, consistent with infarcts. Conclusion: The increased levels of AChE and the hearth tissue infiltrative lesions induced by the aqueous seed kernel extract of T. peruviana explains in part the poisoning caused by this plant, which can be related to an inflammatory process. PMID:24914300

  12. C1 esterase inhibitor

    MedlinePlus

    ... algorithm for the diagnosis, therapy and management of hereditary angioedema. Allergy Asthma Clin Immunol . 2010;6:24. PMID: ... chap 6. Read More Cirrhosis Complement Glomerulonephritis Hepatitis Hereditary angioedema Kidney transplant Lupus nephritis Systemic lupus erythematosus Ulcerative ...

  13. Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism.

    PubMed

    Bloch, R J

    1979-09-01

    The effects of energy metabolism inhibitors on the distribution of acetylcholine receptors (AChRs) in the surface membranes of non-innervated, cultured rat myotubes were studied by visualizing the AChRs with monotetramethylrhodamine-alpha-bungarotoxin. Incubation of myotubes with inhibitors of energy metabolism causes a large decrease in the fraction of myotubes displaying clusters of AChR. This decrease is reversible, and is dependent on temperature, the concentration of inhibitor, and the duration of treatment. Cluster dispersal is probably not the result of secondary effects on Ca++ or cyclic nucleotide metabolism, membrane potential, cytoskeletal elements, or protein synthesis. Sequential observations of identified cells treated with sodium azide showed that clusters appear to disperse by movements of receptors within the sarcolemma without accompanying changes in cell shape. AChR clusters dispersed by pretreating cells with sodium azide rapidly reform upon removal of the inhibitor. Reclustering involves the formation of small aggregates of AChR, which act as foci for further aggregation and which appear to be precursors of large AChR clusters. Small AChR aggregates also appear to be precursors of clusters which form on myotubes never exposed to azide. Reclustering after azide treatment does not necessarily occur at the same sites occupied by clusters before dispersal, nor does it employ only receptors which had previously been in clusters. Cluster reformation can be blocked by cycloheximide, colchicine, and drugs which alter the intracellular cation composition.

  14. Inactivation of arginine esterase E-I of Bitis gabonica venom by irreversible inhibitors including a water-soluble carbodiimide, a chloromethyl ketone and isatoic anhydride.

    PubMed

    Gravett, P S; Viljoen, C C; Oosthuizen, M M

    1991-01-01

    1. Esterase E-I from Bitis gabonica was inactivated with irreversible inhibitors which included studies with a water-soluble carbodiimide, an affinity labelling peptide and a mechanism-based inactivator. 2. The reaction with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide was biphasic and the dominant part followed saturation kinetics. At pH 5.5 a rate constant of 0.4 min-1 for inactive enzyme formation was calculated and a dissociation constant (Ki) of 0.2 M for the enzyme-inhibitor complex. 3. Inactivation with D-Phe-Pro-Arg-chloromethyl ketone indicated a two-step mechanism, for which the reaction parameters at pH 8.0 were determined. The Ki value was 0.2 microM and the inactivation rate was 2.5 min-1. 4. With isatoic anhydride pseudo-first-order kinetics was observed. At pH 8.0 a rate constant of 0.9 min-1 and a Ki of 2.0 mM were obtained. The inactivation of the enzyme was found to be governed by a group in the enzyme showing a pK value of 7.3.

  15. The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

    SciTech Connect

    Grandič, Marjana; Aráoz, Romulo; Molgó, Jordi; Turk, Tom; Sepčić, Kristina; Benoit, Evelyne; Frangež, Robert

    2012-12-01

    APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC{sub 50} = 0.74 μM), without affecting directly-elicited twitch tension up to 2.72 μM. The compound (0.007–3.40 μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 μM, without affecting their frequency. Full size endplate potentials, recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC{sub 50} = 0.36 μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1{sub 2}β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC{sub 50} = 0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1{sub 2}β1γδ) than for the mouse (α1{sub 2}β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction. -- Highlights: ► APS12-2 produces concentration-dependent inhibition of nerve-evoked muscle contraction in vitro. ► APS12-2 blocks MEPPs and EPPs at the neuromuscular junction. APS12-2 blocks ACh-activated current in Xenopus oocytes incorporated with Torpedo nAChRs.

  16. Plasma-Derived C1 Esterase Inhibitor for Acute Antibody-Mediated Rejection Following Kidney Transplantation: Results of a Randomized Double-Blind Placebo-Controlled Pilot Study.

    PubMed

    Montgomery, R A; Orandi, B J; Racusen, L; Jackson, A M; Garonzik-Wang, J M; Shah, T; Woodle, E S; Sommerer, C; Fitts, D; Rockich, K; Zhang, P; Uknis, M E

    2016-05-16

    Antibody-mediated rejection (AMR) is typically treated with plasmapheresis (PP) and intravenous immunoglobulin (standard of care; SOC); however, there is an unmet need for more effective therapy. We report a phase 2b, multicenter double-blind randomized placebo-controlled pilot study to evaluate the use of human plasma-derived C1 esterase inhibitor (C1 INH) as add-on therapy to SOC for AMR. Eighteen patients received 20 000 units of C1 INH or placebo (C1 INH n = 9, placebo n = 9) in divided doses every other day for 2 weeks. No discontinuations, graft losses, deaths, or study drug-related serious adverse events occurred. While the study's primary end point, a difference between groups in day 20 pathology or graft survival, was not achieved, the C1 INH group demonstrated a trend toward sustained improvement in renal function. Six-month biopsies performed in 14 subjects (C1 INH = 7, placebo = 7) showed no transplant glomerulopathy (TG) (PTC+cg≥1b) in the C1 INH group, whereas 3 of 7 placebo subjects had TG. Endogenous C1 INH measured before and after PP demonstrated decreased functional C1 INH serum concentration by 43.3% (p < 0.05) for both cohorts (C1 INH and placebo) associated with PP, although exogenous C1 INH-treated patients achieved supraphysiological levels throughout. This new finding suggests that C1 INH replacement may be useful in the treatment of AMR.

  17. Model-based evaluation of similarity in pharmacokinetics of two formulations of the blood-derived plasma product c1 esterase inhibitor.

    PubMed

    Keizer, Ron J; Budde, Ilona Kleine; Sprengers, Paul F W; Levi, M; Beijnen, Jos H; Huitema, Alwin D R

    2012-02-01

    A novel formulation of C1 esterase inhibitor concentrate, a plasma product used in the treatment of hereditary angioedema (HAE), was studied in a clinical trial for similarity in pharmacokinetics (PK) compared with the reference product. Direct trial data were limited given the availability of patients, and therefore a modeling approach was used to study similarity. Type I error of the study was evaluated using simulations based on retrospective data. A population PK modeling analysis was performed on data from the trial. Analysis of variance was carried out on results of a noncompartmental PK analysis (NCA) of the clinical data. Simulations showed that type I error was inflated to 62% (P < .05) when bioequivalence criteria (confidence intervals within 80%-125%) were adhered to strictly. In the clinical trial, 13 HAE patients were evaluable. The population PK analysis showed no significant differences in PK parameters, whereas confidence intervals for all parameters were within 80% to 125%. The relative differences in area under the curve, incremental recovery, and mean residence time estimated using NCA were all close to 1. The novel formulation showed similar PK characteristics to the original formulation. The model-based approach showed that strict criteria for PK comparison could not be applied in this analysis.

  18. Reactivity versus steric effects in fluorinated ketones as esterase inhibitors: a quantum mechanical and molecular dynamics study

    PubMed Central

    Rayo, Josep; Muñoz, Lourdes; Rosell, Gloria; Hammock, Bruce D.; Guerrero, Angel

    2010-01-01

    Carboxylesterases (CEs) are a family of ubiquitous enzymes with broad substrate specificity, and their inhibition may have important implications in pharmaceutical and agrochemical fields. One of the most potent inhibitors both for mammalian and insect CEs are trifluoromethyl ketones (TFMKs), but the mechanism of action of these chemicals is not completely understood. This study examines the balance between reactivity versus steric effects in modulating the activity against human carboxylesterase 1. The intrinsic reactivity of the ketone moiety is determined from quantum mechanical computations, which combine gas phase B3LYP calculations with hydration free energies estimated with the IEF/MST model. In addition, docking and molecular dynamics simulations are used to explore the binding mode of the inhibitors along the deep gorge that delineates the binding site. The results point out that the activity largely depends on the nature of the fluorinated ketone, since the activity is modulated by the balance between the intrinsic electrophilicity of the carbonyl carbon atom and the ratio between keto and hydrate forms. However, the results also suggest that the correct alignment of the alkyl chain in the binding site can exert a large influence on the inhibitory activity, as this effect seems to override the intrinsic reactivity features of the fluorinated ketone. Overall, the results sustain a subtle balance between reactivity and steric effects in modulating the inhibitory activity of TFMK inhibitors. PMID:20676708

  19. NSC23766, a widely used inhibitor of Rac1 activation, additionally acts as a competitive antagonist at muscarinic acetylcholine receptors.

    PubMed

    Levay, Magdolna; Krobert, Kurt Allen; Wittig, Karola; Voigt, Niels; Bermudez, Marcel; Wolber, Gerhard; Dobrev, Dobromir; Levy, Finn Olav; Wieland, Thomas

    2013-10-01

    Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N(6)-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M2 muscarinic acetylcholine receptor (M2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly Giβγ-mediated, adenosine-induced activation of Gi protein-regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K(+) current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A1 receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca(2+) concentrations in human embryonic kidney 293 (HEK-293) cells expressing M1, M2, or M3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M2 and M3 mAChR crystal

  20. Plasma-derived human C1-esterase inhibitor does not prevent mechanical ventilation-induced pulmonary complement activation in a rat model of Streptococcus pneumoniae pneumonia.

    PubMed

    de Beer, F M; Aslami, H; Hoeksma, J; van Mierlo, G; Wouters, D; Zeerleder, S; Roelofs, J J T H; Juffermans, N P; Schultz, M J; Lagrand, W K

    2014-11-01

    Mechanical ventilation has the potential to cause lung injury, and the role of complement activation herein is uncertain. We hypothesized that inhibition of the complement cascade by administration of plasma-derived human C1-esterase inhibitor (C1-INH) prevents ventilation-induced pulmonary complement activation, and as such attenuates lung inflammation and lung injury in a rat model of Streptococcus pneumoniae pneumonia. Forty hours after intratracheal challenge with S. pneumoniae causing pneumonia rats were subjected to ventilation with lower tidal volumes and positive end-expiratory pressure (PEEP) or high tidal volumes without PEEP, after an intravenous bolus of C1-INH (200 U/kg) or placebo (saline). After 4 h of ventilation blood, broncho-alveolar lavage fluid and lung tissue were collected. Non-ventilated rats with S. pneumoniae pneumonia served as controls. While ventilation with lower tidal volumes and PEEP slightly amplified pneumonia-induced complement activation in the lungs, ventilation with higher tidal volumes without PEEP augmented local complement activation more strongly. Systemic pre-treatment with C1-INH, however, failed to alter ventilation-induced complement activation with both ventilation strategies. In accordance, lung inflammation and lung injury were not affected by pre-treatment with C1-INH, neither in rats ventilated with lower tidal volumes and PEEP, nor rats ventilated with high tidal volumes without PEEP. Ventilation augments pulmonary complement activation in a rat model of S. pneumoniae pneumonia. Systemic administration of C1-INH, however, does not attenuate ventilation-induced complement activation, lung inflammation, and lung injury.

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

    PubMed

    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.

  2. Esterase isozymes of the hen's oviduct.

    PubMed

    Grunder, A A; Holland, K G

    1977-11-01

    Esterase isozymes of magnum, isthmus and uterus of three strains of Single Comb White Leghorn hens were examined by zone electrophoresis on starch gels. Although three regions (I, II and III) of esterase activity were observed, the electrophoretic system was optimized to characterize the pattern of up to five zones of esterase activity that were identified in Region I. These esterases were classified as aliesterases based on reactions in the presence of various substrates and inhibitors. No genetic polymorphisms were observed for these isozymes. However, two of these isozymes were perceived to have an electrophoretic mobility slightly faster in patterns of the magnum of layers than in the isthmus, uterus, and magnum of non-layers. It was shown that egg albumen was present in relatively high quantities in the magnum of layers and that egg albumen, when added to supernatant preparations of isthmus, uterus and magnum of non-layers, caused the faster electrophoretic mobility of these two esterase isozymes. No relation between specific gravity of eggs laid by hens and presence of various Region I esterase isozymes could be detected.

  3. Antithrombin III, but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting.

    PubMed

    Kellner, Patrick; Nestler, Frank; Leimert, Anja; Bucher, Michael; Czeslick, Elke; Sablotzki, Armin; Raspè, Christoph

    2014-12-01

    In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting. Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27±7years) were pre-incubated with clinically relevant concentrations of AT-III (n=11) and C1-INH (n=12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n=6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes. This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs. Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1

  4. Feasibility of home infusion and self-administration of nanofiltered C1 esterase inhibitor for routine prophylaxis in patients with hereditary angioedema and characterization of a training and support program.

    PubMed

    Gregory, Cherylann; Landmesser, Ladonna M; Corrigan, Larry; Mariano, David

    2014-01-01

    Hereditary angioedema (HAE) is a rare, chronic disease of C1 inhibitor deficiency. Study researchers evaluated the prevalence of home and self-administration of nanofiltered, human-derived C1 esterase inhibitor infusions and the implementation of a nursing training and support program. Home administration rate increased from 49.0% to 75.8%. The percentage who self-administered increased from 20.3% to 43.9%. Doses per week averaged 1.85 at home compared with 1.40 in infusion centers and physicians' offices. Patients required an average of 5 visits to be trained. Self-administration is a viable, feasible option in the management of HAE, which is facilitated by a nurse-managed training and support program.

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

    PubMed

    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

  6. Leukocyte esterase urine test

    MedlinePlus

    ... the urine. This may mean you have a urinary tract infection . If this test is positive, the urine should ... Results Mean An abnormal result indicates a possible urinary tract infection. Alternative Names WBC esterase Images Male urinary system ...

  7. pH-dependent hydrolysis of acetylcholine: Consequences for non-neuronal acetylcholine.

    PubMed

    Wessler, Ignaz; Michel-Schmidt, Rosmarie; Kirkpatrick, Charles James

    2015-11-01

    Acetylcholine is inactivated by acetylcholinesterase and butyrylcholinesterase and thereby its cellular signalling is stopped. One distinguishing difference between the neuronal and non-neuronal cholinergic system is the high expression level of the esterase activity within the former and a considerably lower level within the latter system. Thus, any situation which limits the activity of both esterases will affect the non-neuronal cholinergic system to a much greater extent than the neuronal one. Both esterases are pH-dependent with an optimum at pH above 7, whereas at pH values below 6 particularly the specific acetylcholinesterase is more or less inactive. Thus, acetylcholine is prevented from hydrolysis at such low pH values. The pH of the surface of the human skin is around 5 and therefore non-neuronal acetylcholine released from keratinocytes can be detected in a non-invasive manner. Several clinical conditions like metabolic acidosis, inflammation, fracture-related haematomas, cardiac ischemia and malignant tumours are associated with local or systemic pH values below 7. Thus, the present article describes some consequences of an impaired inactivation of extracellular non-neuronal acetylcholine.

  8. Evaluating Prodrug Strategies for Esterase-Triggered Release of Alcohols

    PubMed Central

    Perez, Christian; Daniel, Kevin B.

    2013-01-01

    Prodrugs are effective tools in overcoming drawbacks typically associated with drug formulation and delivery. Those employing esterase-triggered functional groups are frequently utilized to mask polar carboxylic acids and phenols, increasing drug-like properties such as lipophilicity. Herein we detail a comprehensive assessment for strategies that effectively release hydroxyl and phenolic moieties in the presence of an esterase. Matrix metalloproteinases (MMPs) serve as our proof-of-concept target. Three distinct ester-responsive protecting groups are incorporated into MMP proinhibitors containing hydroxyl moieties. Analytical evaluation of the proinhibitors demonstrates that the use of a benzyl ether group appended to the esterase trigger leads to considerably faster kinetics of conversion and enhanced aqueous stability when compared to more conventional approaches where the trigger is directly attached to the inhibitor. Biological assays confirm that all protecting groups effectively cleave in the presence of esterase to generate the active inhibitor. PMID:23929690

  9. Brain regional acetylcholinesterase activity and muscarinic acetylcholine receptors in rats after repeated administration of cholinesterase inhibitors and its withdrawal

    SciTech Connect

    Kobayashi, Haruo . E-mail: hk1664@iwate-u.ac.jp; Suzuki, Tadahiko; Sakamoto, Maki; Hashimoto, Wataru; Kashiwada, Keiko; Sato, Itaru; Akahori, Fumiaki; Satoh, Tetsuo

    2007-03-15

    Activity of acetylcholinesterase (AChE) and specific binding of [{sup 3}H]quinuclidinyl benzilate (QNB), [{sup 3}H]pirenzepine (PZP) and [{sup 3}H]AF-DX 384 to muscarinic acetylcholine receptor (mAChR) preparations in the striatum, hippocampus and cortex of rats were determined 1, 6 and 11 days after the last treatment with an organophosphate DDVP, a carbamate propoxur or a muscarinic agonist oxotremorine as a reference for 7 and 14 days. AChE activity was markedly decreased in the three regions 1 day after the treatment with DDVP for 7 and 14 days with a gradual recovery 6 to 11 days, and much less decreased 1, 6 and 11 days after the treatment with propoxur for 7 days but not for 14 days in the hippocampus and cortex. The binding of [{sup 3}H]-QNB, PZP and AF-DX 384 in the three regions was generally decreased by the treatment with DDVP for 7 and 14 days. Such down-regulations were generally restored 6 or 11 days after the treatment for 7 but not for 14 days. The down-regulation or up-regulation as measured by [{sup 3}H]-QNB, PZP and AF-DX 384 was observed 1, 6 or 11 days after treatment with propoxur for 7 days and/or 14 days. Repeated treatment with oxotremorine produced similar effects except AChE activity to DDVP. These results suggest that repeated inhibition of AChE activity may usually cause down-regulation of mAChRs with some exception in the hippocampus when a reversible antiChE propoxur is injected.

  10. Organophosphates and monocyte esterase deficiency.

    PubMed Central

    McClean, E; Mackey, H; Markey, G M; Morris, T C

    1995-01-01

    AIMS--To examine the possibility that monocyte esterase deficiency (MED) could be caused by exposure to organophosphates. METHODS--Pseudocholinesterase, paraoxonase and arylesterase activities were measured in the serum and acetylcholinesterase activity was measured in the red cells of a group of monocyte esterase deficient subjects and compared with the enzyme activities of a control group of monocyte esterase positive subjects. RESULTS--No significant difference was found between the enzyme activities of the monocyte esterase deficient group and the control group for any of the esterases investigated. CONCLUSION--Current or recent exposure to organophosphorus is not the cause of MED. PMID:7560207

  11. Esterase mutation is a mechanism of resistance to antimalarial compounds

    PubMed Central

    Istvan, Eva S.; Mallari, Jeremy P.; Corey, Victoria C.; Dharia, Neekesh V.; Marshall, Garland R.; Winzeler, Elizabeth A.; Goldberg, Daniel E.

    2017-01-01

    Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance. PMID:28106035

  12. Simultaneous determination of curcumin diethyl disuccinate and its active metabolite curcumin in rat plasma by LC-MS/MS: Application of esterase inhibitors in the stabilization of an ester-containing prodrug.

    PubMed

    Ratnatilaka Na Bhuket, Pahweenvaj; Niwattisaiwong, Nuansri; Limpikirati, Patanachai; Khemawoot, Phisit; Towiwat, Pasarapa; Ongpipattanakul, Boonsri; Rojsitthisak, Pornchai

    2016-10-15

    Four esterase inhibitors, ethylenediamine tetraacetic acid disodium (Na2EDTA), sodium fluoride (NaF), bis(4-nitrophenyl) phosphate (BNPP) and phenylmethanesulfonyl fluoride (PMSF), were evaluated for their inhibitory effects on enzymatic hydrolysis of labile phenolate esters in curcumin diethyl disuccinate (CDD), a prodrug of curcumin (CUR), in rat plasma. BNPP and PMSF at 10mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10mM was subsequently selected to prevent ex vivo metabolism of CDD throughout LC-MS/MS analysis of CDD and CUR in rat plasma. A simple protein precipitation technique using acetonitrile as a precipitating agent was used to extract CDD, CUR and dimethylcurcumin (DMC), an internal standard, from rat plasma. Chromatographic separation was performed on a Halo C8 column (4.6×50mm, 2.7μm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27v/v) with a flow rate of 0.4mLmin(-1). An AB SCIEX QTRAP(®) 6500 mass spectrometer was operated using a positive ion electrospray mode for ionization and detection of analytes and internal standard. Calibration curves for CDD and CUR were established using 50μL of rat plasma over the concentration range of 1-500ngmL(-1). The developed method was fully validated according to US Food and Drug Administration (FDA) guidelines for selectivity, sensitivity, linearity, accuracy, precision, dilution integrity, recovery, matrix effect, and stability. The validated method was applied to evaluate the pharmacokinetics of CDD and CUR in rats after a single intravenous dose of 40mgkg(-1). The method using BNPP as an esterase inhibitor was successful in determining the remaining CDD in rat plasma. The pharmacokinetic results indicate that CDD in rats is converted instantaneously to CUR after intravenous administration and a higher CUR plasma concentration at 5min is achieved in comparison with direct intravenous injection of CUR.

  13. Binding sites for. alpha. -bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the. alpha. -subunit of the nicotinic acetylcholine receptor

    SciTech Connect

    Donnelly-Roberts, D.L.; Lentz, T.L. )

    1991-07-30

    The binding of the competitive antagonist {alpha}-bungarotoxin ({alpha}-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the {alpha}-subunit of the Torpedo acetylcholine receptor has been characterized. {sup 125}I-{alpha}-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by {alpha}-Btx d-tubocurarine and NaCl. In the presence of 0.02% sodium dodecyl sulfate, {sup 125}I-{alpha}-Btx bound to the 56-residue peptide with a K{sub D} of 3.5 nM, as determined by equilibrium saturation binding studies. Because {alpha}Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, ({sup 3}H)PCP was bound to the 172-227 peptide. ({sup 3}H)PCP binding was inhibited by chlorpromazine, tetracaine, and dibucaine. It is concluded that a high-affinity binding site for PCP is located between residues 205 and 227, which includes the first 18 residues of transmembrane segment M1, and that a low-affinity site is located in the competitive antagonist binding site between residues 173 and 204. These results show that a synthetic peptide comprising residues 172-227 of the {alpha} subunit contains three binding sites, one for {alpha}-Btx and two for PCP. Previous studies on the intact receptor indicate high-affinity PCP binding occurs in the receptor channel.

  14. Successful use of daily intravenous infusion of C1 esterase inhibitor concentrate in the treatment of a hereditary angioedema patient with ascites, hypovolemic shock, sepsis, renal and respiratory failure.

    PubMed

    Pham, Hoang; Santucci, Stephanie; Yang, William H

    2014-01-01

    Hereditary angioedema (HAE) is a rare autosomal dominant disease most commonly associated with defects in C1 esterase inhibitor (C1-INH). HAE manifests as recurrent episodes of edema in various body locations. Atypical symptoms, such as ascites, acute respiratory distress syndrome, and hypovolemic shock, have also been reported. Management of HAE conventionally involves the treatment of acute attacks, as well as short- and long-term prophylaxis. Since attacks can be triggered by several factors, including stress and physical trauma, prophylactic therapy is recommended for patients undergoing surgery. Human plasma-derived C1-INH (pdC1-INH) concentrate is indicated for the treatment of both acute HAE attacks and pre-procedure prevention of HAE episodes in patients undergoing medical, dental, or surgical procedures. We report the first case of a patient with HAE who experienced an abdominal attack precipitated by a retroperitoneal bleed while being converted from warfarin to heparin in preparation for surgery. Subsequently, the patient had a protracted course in hospital with other complications, which included hypovolemic shock, ascites, severe sepsis from nosocomial pneumonia, renal and respiratory failure. Despite intensive interventions, the patient remained in a critical state for months; however, after a trial of daily intravenous infusion of pdC1-INH concentrate (Berinert®, CSL Behring GmbH, Marburg, Germany), clinical status improved, particularly renal function. Therefore, pdC1-INH concentrate may be an effective treatment option to consider for critically-ill patients with HAE.

  15. Phenyl valerate esterase activity of human butyrylcholinesterase.

    PubMed

    Mangas, Iris; Vilanova, Eugenio; Estévez, Jorge

    2017-03-15

    Phenyl valerate is used for detecting and measuring neuropathy target esterase (NTE) and has been used for discriminating esterases as potential target in hen model of organophosphorus delayed neuropathy. In previous studies we observed that phenyl valerate esterase (PVase) activity of an enzymatic fraction in chicken brain might be due to a butyrylcholinesterase protein (BuChE), and it was suggested that this enzymatic fraction could be related to the potentiation/promotion phenomenon of the organophosphate-induced delayed neuropathy (OPIDN). In this work, PVase activity of purified human butyrylcholinesterase (hBuChE) is demonstrated and confirms the novel observation that a relationship of BuChE with PVase activities is also relevant for humans, as is, therefore the potential role in toxicity for humans. The KM and catalytic constant (kcat) were estimated as 0.52/0.72 µM and 45,900/49,200 min(-1) respectively. Furthermore, this work studies the inhibition by preincubation of PVase and cholinesterase activities of hBuChE with irreversible inhibitors (mipafox, iso-OMPA or PMSF), showing that these inhibitors interact similarly in both activities with similar second-order inhibition constants. Acethylthiocholine and phenyl valerate partly inhibit PVase and cholinesterase activities, respectively. All these observations suggest that both activities occur in the same active center. The interaction with a reversible inhibitor (ethopropazine) showed that the cholinesterase activity was more sensitive than the PVase activity, showing that the sensitivity for this reversible inhibitor is affected by the nature of the substrate. The present work definitively establishes the capacity of BuChE to hydrolyze the carboxylester phenyl valerate using a purified enzyme (hBuChE). Therefore, BuChE should be considered in the research of organophosphorus targets of toxicity related with PVase proteins.

  16. Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms.

    PubMed

    Abongwa, Melanie; Buxton, Samuel K; Robertson, Alan P; Martin, Richard J

    2016-01-01

    Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex

  17. Recent developments in the treatment of acute abdominal and facial attacks of hereditary angioedema: focus on human C1 esterase inhibitor

    PubMed Central

    Cardona, Lourdes Pastó; Bellfill, Ramon Lleonart; Caus, Joaquim Marcoval

    2010-01-01

    Hereditary angioedema (HAE) is a potentially fatal genetic disorder typified by a deficiency (type I) or dysfunction (type II) of the C1-inhibitor (C1-INH) and characterized by swelling of the extremities, face, trunk, abdominal viscera, and upper airway. Type III is normal estrogen-sensitive C1-INH HAE. Bradykinin, the main mediator of HAE, binds to endothelial B2 receptors, increasing vascular permeability and resulting in edema. HAE management includes short- and long-term prophylaxis. For treating acute episodes, C1-INH concentrate is recommended with regression of symptoms achieved in 30–90 min. Infusions of 500–1000 U have been used in Europe for years. Two plasma-derived C1-INH concentrates have been licensed recently in the United States: Berinert® for treating acute attacks and Cinryze® for prophylaxis in adolescent/adult patients. A recombinant C1-INH that is being considered for approval (conestat alfa) exhibited significant superiority versus placebo. Ecallantide (Kalbitor®) is a selective kallikrein inhibitor recently licensed in the United States for treating acute attacks in patients aged >16 years. It is administered in three 10-mg subcutaneous injections with the risk of anaphylactic reactions. Icatibant (Firazyr®) is a bradykinin B2 receptor competitor. It is administered subcutaneously as a 30-mg injection and approved in Europe but not in the United States. PMID:23776358

  18. New insights on molecular interactions of organophosphorus pesticides with esterases.

    PubMed

    Mangas, Iris; Estevez, Jorge; Vilanova, Eugenio; França, Tanos Celmar Costa

    2017-02-01

    Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the

  19. MECHANISMS OF ACTIVATION OF C'1 ESTERASE IN HEREDITARY ANGIONEUROTIC EDEMA PLASMA IN VITRO

    PubMed Central

    Donaldson, Virginia H.

    1968-01-01

    The generation of C'1 esterase activity in siliconed plasma obtained from individuals with hereditary angioneurotic edema in remission tends to occur spontaneously, but can be hastened during its incubation with preparations of activated Hageman factor. This effect of activated Hageman factor could not be shown during its incubation with normal siliconed plasma, nor could consumption of normal serum inhibition of C'1 esterase be clearly shown. Soy bean trypsin inhibitor and heparin could impair this enhanced generation of C'1 esterase but neither inhibits the esterolytic function of C'1 esterase once formed. Trasylol was less effective in blocking this effect of activated Hageman factor. While the mechanism of the effect of activated Hageman factor upon C'1 activation remains obscure, it is apparent that some intermediate steps, possibly involving a kinin-forming system of plasma, may play a role. PMID:5299945

  20. Influenza C virus esterase: analysis of catalytic site, inhibition, and possible function

    SciTech Connect

    Vlasak, R.; Muster, T.; Lauro, A.M.; Powers, J.C.; Palese, P.

    1989-05-01

    The active site serine of the acetylesterase of influenza C virus was localized to amino acid 71 of the hemagglutinin-esterase protein by affinity labeling with /sup 3/H-labeled diisopropylfluorophosphate. This serine and the adjacent amino acids (Phe-Gly-Asp-Ser) are part of a consensus sequence motif found in serine hydrolases. Since comparative analysis failed to reveal esterase sequence similarities with other serine hydrolases, the authors suggest that this viral enzyme is a serine hydrolase constituting a new family of serine esterases. Furthermore, they found that the influenza C virus esterase was inhibited by isocoumarin derivatives, with 3,4-dichloroisocoumarin being the most potent inhibitor. Addition of this compound prevented elution of influenza C virus from erythrocytes and inhibited virus infectivity, possibly through inhibition of virus entry into cells.

  1. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

  2. Progesterone Modulates a Neuronal Nicotinic Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Valera, S.; Ballivet, M.; Bertrand, D.

    1992-10-01

    The major brain nicotinic acetylcholine receptor is assembled from two subunits termed α 4 and nα 1. When expressed in Xenopus oocytes, these subunits reconstitute a functional acetylcholine receptor that is inhibited by progesterone levels similar to those found in serum. In this report, we show that the steroid interacts with a site located on the extracellular part of the protein, thus confirming that inhibition by progesterone is not due to a nonspecific perturbation of the membrane bilayer or to the activation of second messengers. Because inhibition by progesterone does not require the presence of agonist, is voltage-independent, and does not alter receptor desensitization, we conclude that the steroid is not an open channel blocker. In addition, we show that progesterone is not a competitive inhibitor but may interact with the acetylcholine binding site and that its effect is independent of the ionic permeability of the receptor.

  3. The search of the target of promotion: Phenylbenzoate esterase activities in hen peripheral nerve

    SciTech Connect

    Moretto, A. . E-mail: angelo.moretto@icps.it; Nicolli, A.; Lotti, M.

    2007-03-15

    Certain esterase inhibitors, such as carbamates, phosphinates and sulfonyl halides, do not cause neuropathy as some organophosphates, but they may exacerbate chemical or traumatic insults to axons. This phenomenon is called promotion of axonopathies. Given the biochemical and toxicological characteristics of these compounds, the hypothesis was made that the target of promotion is a phenyl valerate (PV) esterase similar to neuropathy target esterase (NTE), the target of organophosphate induced delayed polyneuropathy. However, attempts to identify a PV esterase in hen peripheral nerve have been, so far, unsuccessful. We tested several esters, other than PV, as substrates of esterases from crude homogenate of the hen peripheral nerve. The ideal substrate should be poorly hydrolysed by NTE but extensively by enzyme(s) that are insensitive to non-promoters, such as mipafox, and sensitive to promoters, such as phenyl methane sulfonyl fluoride (PMSF). When phenyl benzoate (PB) was used as substrate, about 65% of total activity was resistant to the non-promoter mipafox (up to 0.5 mM, 20 min, pH 8.0), that inhibits NTE and other esterases. More than 90% of this resistant activity was sensitive to the classical promoter PMSF (1 mM, 20 min, pH 8.0) with an IC{sub 50} of about 0.08 mM (20 min, pH 8.0). On the contrary, the non-promoter p-toluene sulfonyl fluoride caused only about 10% inhibition at 0.5 mM. Several esterase inhibitors including, paraoxon, phenyl benzyl carbamate, di-n-butyl dichlorovinyl phosphate and di-isopropyl fluorophosphate, were tested both in vitro and in vivo for inhibition of this PB activity. Mipafox-resistant PMSF-sensitive PB esterase activity(ies) was inhibited by promoters but not by non promoters and neuropathic compounds.

  4. Effect of halogenated benzenes on acetanilide esterase, acetanilide hydroxylase and procaine esterase in rats.

    PubMed

    Carlson, G P; Dziezak, J D; Johnson, K M

    1979-07-01

    1,2,4-Trichlorobenzene, 1,3,5-trichlorobenzene, hexachlorobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene and hexabromobenzene were compared for their abilities to induce acetanilide esterase, acentailide hydroxylase and procaine esterase. Except for hexabromobenzene all induced acetanilide esterase whereas the hydroxylation of acetanilide was seen only with the fully halogenated benzenes and with 1,3,5-tribromobenzene. Hepatic procaine esterase activity was increased by the three chlorinated benzenes and 1,2,4-tribromobenzene.

  5. Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells.

    PubMed

    Wawrzeńczyk, A; Nałecz, K A; Nałecz, M J

    1995-06-01

    Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 microM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of beta-oxidation might be correlated with the lower level of acetylcholine synthesis.

  6. Esterase profiles of organophosphorus compounds in vitro predict their behavior in vivo.

    PubMed

    Makhaeva, Galina F; Rudakova, Elena V; Serebryakova, Olga G; Aksinenko, Alexey Yu; Lushchekina, Sofya V; Bachurin, Sergey O; Richardson, Rudy J

    2016-11-25

    We studied 4 serine esterases (EOHs) that are associated with the following consequences from their inhibition by organophosphorus compounds (OPCs): acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE; inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity, OPIDN). The relative degree of inhibition of these EOHs constitutes the "esterase profile" of an OPC, which we hypothesize can serve as a predictor of its overall physiological effects. To test this hypothesis, we selected 3 OPCs known from previous work on reference enzymes to span a wide range of esterase profiles, neuropathic potential, and acute cholinergic toxicity. For each compound, we determined in vitro IC50 and in vivo ED50 values for inhibition of AChE, BChE, CaE, and NTE in mouse brain and blood. The results showed good correlations between in vitro and in vivo measures of potency and selectivity except for brain CaE, a tissue-specific isoform of the enzyme that was less sensitive to the test compounds than expected. Thus, this synthesis of new and previously published results indicates that the concept of the esterase profile of OPCs is useful for the prediction of therapeutic and toxic effects in vivo.

  7. Mechanism and diversity of the erythromycin esterase family of enzymes.

    PubMed

    Morar, Mariya; Pengelly, Kate; Koteva, Kalinka; Wright, Gerard D

    2012-02-28

    Macrolide antibiotics such as azithromycin and erythromycin are mainstays of modern antibacterial chemotherapy, and like all antibiotics, they are vulnerable to resistance. One mechanism of macrolide resistance is via drug inactivation: enzymatic hydrolysis of the macrolactone ring catalyzed by erythromycin esterases, EreA and EreB. A genomic enzymology approach was taken to gain insight into the catalytic mechanisms and origins of Ere enzymes. Our analysis reveals that erythromycin esterases comprise a separate group in the hydrolase superfamily, which includes homologues of uncharacterized function found on the chromosome of Bacillus cereus, Bcr135 and Bcr136, whose three-dimensional structures have been determined. Biochemical characterization of Bcr136 confirms that it is an esterase that is, however, unable to inactivate macrolides. Using steady-state kinetics, homology-based structure modeling, site-directed mutagenesis, solvent isotope effect studies, pH, and inhibitor profiling performed in various combinations for EreA, EreB, and Bcr136 enzymes, we identified the active site and gained insight into some catalytic features of this novel enzyme superfamily. We rule out the possibility of a Ser/Thr nucleophile and show that one histidine, H46 (EreB numbering), is essential for catalytic function. This residue is proposed to serve as a general base in activation of a water molecule as the reaction nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are distinct, with only EreA inhibited by chelating agents and hypothesized to contain a noncatalytic metal. Detailed characterization of these esterases allows for a direct comparison of the resistance determinants, EreA and EreB, with their prototype, Bcr136, and for the discussion of their potential connections.

  8. Characterization and affinity purification of juvenile hormone esterase from Bombyx mori.

    PubMed

    Shiotsuki, T; Bonning, B C; Hirai, M; Kikuchi, K; Hammock, B D

    2000-08-01

    Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.

  9. Distinction between 'A'-esterases and arylesterases. Implications for esterase classification.

    PubMed Central

    Mackness, M I; Thompson, H M; Hardy, A R; Walker, C H

    1987-01-01

    'A'-esterase activities (substrates paraoxon and pirimiphos-methyloxon) and arylesterase activities (substrate phenyl acetate) were assayed in the sera of 14 species of birds representing seven different orders and 11 species of mammal representing five different orders. Ten species of birds had no detectable 'A'-esterase, and the remaining four species only low activity, yet all birds showed considerable arylesterase activity (16.8-99.3 mumol/min per ml of serum). Ten species of mammal showed both 'A'- and 'aryl'-esterase activities. In humans, gel filtration of serum completely separated peaks representing paraoxonase and arylesterase activities. Thus, in both birds and humans, serum enzymes exist that express arylesterase activity but not 'A'-esterase activity. These findings suggest that a distinction should be made between these two types of esterase in future classifications. PMID:2822017

  10. Morphogenetic roles of acetylcholine.

    PubMed Central

    Lauder, J M; Schambra, U B

    1999-01-01

    In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive extensive innervation from cholinergic neurons in the basal forebrain, beginning prenatally and continuing throughout the period of active growth and synaptogenesis. Acute exposure to ethanol in early gestation (which prevents formation of basal forebrain cholinergic neurons) or neonatal lesioning of basal forebrain cholinergic neurons, significantly compromises cortical development and produces persistent impairment of cognitive functions. Neonatal visual deprivation alters developmental expression of muscarinic acetylcholine receptors (mAChR) in visual cortex, whereas local infusion of mAChR antagonists impairs plasticity of visual cortical neurons. These findings raise the possibility that exposure to environmental neurotoxins that affect cholinergic systems may seriously compromise brain development and have long-lasting morphologic, neurochemical, and functional consequences. PMID:10229708

  11. Phenolic acid esterases, coding sequences and methods

    DOEpatents

    Blum, David L.; Kataeva, Irina; Li, Xin-Liang; Ljungdahl, Lars G.

    2002-01-01

    Described herein are four phenolic acid esterases, three of which correspond to domains of previously unknown function within bacterial xylanases, from XynY and XynZ of Clostridium thermocellum and from a xylanase of Ruminococcus. The fourth specifically exemplified xylanase is a protein encoded within the genome of Orpinomyces PC-2. The amino acids of these polypeptides and nucleotide sequences encoding them are provided. Recombinant host cells, expression vectors and methods for the recombinant production of phenolic acid esterases are also provided.

  12. Properties of phenyl valerate esterase activities from chicken serum are comparable with soluble esterases of peripheral nerves in relation with organophosphorus compounds inhibition.

    PubMed

    Garcia-Pérez, Adolfo G; Barril, José; Estévez, Jorge; Vilanova, Eugenio

    2003-04-30

    Chicken serum, the usual in vivo animal for testing organophosphorus delayed neuropathy, has long been reported not to contain a homologous activity of the neuronal neuropathy target esterase (NTE) activity when it is assayed according to standard methods as the phenyl valerate esterase (PVase) activity, which is resistant to paraoxon and sensitive to mipafox. However, a PVase activity (1000-1500 nmol/min/ml) can be measured in serum that is extremely sensitive to both paraoxon, a non-neuropathic organophosphorus compound and mipafox, a model neuropathy inducer. The inhibition was time progressive in both cases, suggesting a covalent phosphorilating reaction. The fixed time inhibition curves suggest at least two sensitive components. The IC50 for 30 min, at 37 degrees C are 6 and 51 nM for paraoxon and 4 and 110 nM for mipafox, for every sensitive component. When paraoxon was removed from a serum sample pretreated with the inhibitor, the paraoxon sensitive PVase activity was recovered, in spite of showing a time progressive inhibition suggesting that hydrolytic dephosphorylating reaction recovered at a significant rate. The reactivation of the phosphorylated enzyme could explain that the time progressive inhibitions curves for long time with paraoxon tend to reach a plateau depending on the inhibition concentration. However, with mipafox, the curve approached the same maximal inhibitions at all concentrations as expected for a permanent covalent irreversible phosphorylation, which is coherent with the observations that the activity remained inhibited after removing the inhibitor. Data of serum esterases described in this paper showed similar properties to those previously reported for peripheral nerve soluble phenylvalerate esterase: (1) extremely high sensitivity to paraoxon and mipafox; (2) time progressive kinetic with two sensitive components; (3) recovery of activity after removal of paraoxon; and (4) permanent inhibition with mipafox. These properties of

  13. Acetylcholine Mediates a Slow Synaptic Potential in Hippocampal Pyramidal Cells

    NASA Astrophysics Data System (ADS)

    Cole, A. E.; Nicoll, R. A.

    1983-09-01

    The hippocampal slice preparation was used to study the role of acetylcholine as a synaptic transmitter. Bath-applied acetylcholine had three actions on pyramidal cells: (i) depolarization associated with increased input resistance, (ii) blockade of calcium-activated potassium responses, and (iii) blockade of accommodation of cell discharge. All these actions were reversed by the muscarinic antagonist atropine. Stimulation of sites in the slice known to contain cholinergic fibers mimicked all the actions. Furthermore, these evoked synaptic responses were enhanced by the cholinesterase inhibitor eserine and were blocked by atropine. These findings provide electrophysiological support for the role of acetylcholine as a synaptic transmitter in the brain and demonstrate that nonclassical synaptic responses involving the blockade of membrane conductances exist in the brain.

  14. Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases.

    PubMed

    Tang, J; Chambers, J E

    1999-01-01

    Paraoxon, the active metabolite of parathion, can be detoxified through a noncatalytic pathway by carboxylesterases and a catalytic pathway by calcium-dependent A-esterases, producing p-nitrophenol as a common metabolite. The detoxication patterns of carboxylesterases and A-esterases were investigated in vitro in the present study with a high tissue concentration (75 mg/mL rat liver homogenate or 50% rat serum solution) to more closely reflect enzyme concentrations in intact tissues. A final paraoxon concentration of 3.75 microM was used to incubate with liver homogenates or serum solutions for 5 seconds or 3, 5, 15, or 25 minutes; also 0.625, 1.25, 2.5, 3.125, 3.75, or 5.0 microM paraoxon (final concentration) was incubated with liver homogenates or serum solutions for 15 minutes. Phenyl saligenin cyclic phosphate and EDTA were used to inhibit carboxylesterases and A-esterases, respectively. Significant amounts of p-nitrophenol were generated with or without either inhibitor during a 15 minute incubation with paraoxon from low (0.625 microM) to high (5.0 microM) concentrations. The amount of p-nitrophenol generated via carboxylesterase phosphorylation was greater than via A-esterase-mediated hydrolysis in the initial period of incubation or when incubating with a low concentration of paraoxon. Plateau shape curves of p-nitrophenol concentration versus time or paraoxon concentration indicated that carboxylesterase phosphorylation was saturable. When incubated for long time intervals or with high concentrations of paraoxon, more p-nitrophenol was generated via A-esterase-mediated hydrolysis than from carboxylesterase phosphorylation. The ratio of paraoxon concentration to tissue amount used in in vitro assays of this study was equivalent to dosing a rat with toxicologically relevant dosages. These in vitro data suggest that both carboxylesterases and A-esterases detoxify paraoxon in vivo; carboxylesterases may be an important mode of paraoxon detoxication in initial

  15. New Extremophilic Lipases and Esterases from Metagenomics

    PubMed Central

    López-López, Olalla; Cerdán, Maria E; González Siso, Maria I

    2014-01-01

    Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications. PMID:24588890

  16. Identification of Genes Encoding Microbial Glucuronoyl Esterases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One type of covalent linkages connecting lignin and hemicellulose in plant cell walls is the ester linkage between 4-0-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. An enzyme that could hydrolyze such linkages, named glucuronoyl esterase, occurs in the cellulolytic system of the w...

  17. Phenol esterase activity of porcine skin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The alkyl esters of plant-derived phenols may serve as slow-release sources for cutaneous delivery of antioxidants. The ability of skin esterases to hydrolyze phenolic esters was examined. Esters of tyrosol and hydroxytyrosol were prepared from decanoic and lipoic acids. Ferulic acid was esterified ...

  18. Phenol esterase activity of porcine skin.

    PubMed

    Laszlo, Joseph A; Smith, Leslie J; Evans, Kervin O; Compton, David L

    2015-01-01

    The alkyl esters of plant-derived phenols may serve as slow-release sources for cutaneous delivery of antioxidants. The ability of skin esterases to hydrolyze phenolic esters was examined. Esters of tyrosol and hydroxytyrosol were prepared from decanoic and lipoic acids. Ferulic acid was esterified with octadecanol, glycerol, and dioleoylglycerol. These phenolic derivatives were treated in taurodeoxycholate microemulsion and unilamellar liposomes with ex vivo porcine skin and an aqueous extract of the skin. Extracted esterases hydrolyzed the microemulsions at rates in the order: tyrosyl lipoate > tyrosyl decanoate > hydroxytyrosyl lipoate > hydroxytyrosyl decanoate. The tyrosyl decanoate was subject to comparatively little hydrolysis (10-30% after 24h) when incorporated into liposomes, while hydroxytyrosyl decanoate in liposomes was not hydrolyzed at all by the skin extract. Ferulate esters were not hydrolyzed by the extract in aqueous buffer, microemulsion, nor liposomes. Tyrosyl decanoate applied topically to skin explants in microemulsion were readily hydrolyzed within 4h, while hydrolysis was minimal when applied in liposomes. These findings indicate that porcine skin displays a general esterase activity toward medium-chain esters of tyrosol and hydroxytyrosol, which can be moderated by the physiochemical properties of the lipid vehicle, but no feruloyl esterase activity.

  19. Nonopioid effect of morphine on electrically evoked acetylcholine release from Torpedo electromotor neurons.

    PubMed

    Oron, L; Sarne, Y; Michaelson, D M

    1992-02-01

    The release of acetylcholine from Torpedo electric organ slices following their electrical stimulation was modulated by morphine, by the muscarinic antagonist atropine, and by the nicotinic antagonist tubocurarine. Addition of either atropine or tubocurarine in the presence of the acetylcholinesterase inhibitor phospholine iodide enhanced acetylcholine release. The effects of the two antagonists were additive, a result suggesting that the secreted acetylcholine regulates its own release by activating both muscarinic and nicotinic cholinergic receptors and that these receptors inhibit acetylcholine release by different mechanisms. The effects of opiates on acetylcholine release were examined under conditions in which the cholinergic modulation of release is blocked, i.e., in the presence of atropine and tubocurarine. These experiments revealed that electrically evoked release of acetylcholine is blocked by the opiate agonists morphine and levorphanol. However, the inhibitory effect of morphine on acetylcholine release was not reversed by the opioid antagonist naloxone. Furthermore, dextrorphan, the nonopioid stereoisomer of levorphanol, had the same inhibitory effect as its opioid counterpart. These findings suggest that the effects of opiates on electrically evoked release of acetylcholine are not mediated by opioid receptors. The possible mechanisms underlying these nonopioid effects of morphine and levorphanol are discussed.

  20. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    PubMed

    Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

    2015-03-02

    In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold.

  1. Pseudo-esterase Activity of Human Albumin

    PubMed Central

    Lockridge, Oksana; Xue, Weihua; Gaydess, Andrea; Grigoryan, Hasmik; Ding, Shi-Jian; Schopfer, Lawrence M.; Hinrichs, Steven H.; Masson, Patrick

    2008-01-01

    Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5–6 h there was partial acetylation of 16–17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with β-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 °C was 61 ± 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover. PMID:18577514

  2. Juvenile hormone (JH) esterase of the mosquito Culex quinquefasciatus is not a target of the JH analog insecticide methoprene.

    PubMed

    Kamita, Shizuo G; Samra, Aman I; Liu, Jun-Yan; Cornel, Anthony J; Hammock, Bruce D

    2011-01-01

    Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (k(cat)/K(M) ratio) and V(max) values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE.

  3. Esterase metabolism of cholinesterase inhibitors using rat liver in vitro

    EPA Science Inventory

    A variety of chemicals, such as organophosphate (OP) and carbamate pesticides, nerve agents, and industrial chemicals, inhibit acetylcholinesterase (AChE) leading to overstimulation of the cholinergic nervous system. The resultant neurotoxicity is similar across mammalian species...

  4. Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene, ae1, encoding the acetyl esterase (Ae1) of Hypocrea jecorina was identified by amino terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino ...

  5. Structural analysis of thermostabilizing mutations of cocaine esterase

    SciTech Connect

    Narasimhan, Diwahar; Nance, Mark R.; Gao, Daquan; Ko, Mei-Chuan; Macdonald, Joanne; Tamburi, Patricia; Yoon, Dan; Landry, Donald M.; Woods, James H.; Zhan, Chang-Guo; Tesmer, John J.G.; Sunahara, Roger K.

    2010-09-03

    Cocaine is considered to be the most addictive of all substances of abuse and mediates its effects by inhibiting monoamine transporters, primarily the dopamine transporters. There are currently no small molecules that can be used to combat its toxic and addictive properties, in part because of the difficulty of developing compounds that inhibit cocaine binding without having intrinsic effects on dopamine transport. Most of the effective cocaine inhibitors also display addictive properties. We have recently reported the use of cocaine esterase (CocE) to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality. However, wild-type CocE is relatively unstable at physiological temperatures ({tau}{sub 1/2} {approx} 13 min at 37 C), presenting challenges for its development as a viable therapeutic agent. We applied computational approaches to predict mutations to stabilize CocE and showed that several of these have increased stability both in vitro and in vivo, with the most efficacious mutant (T172R/G173Q) extending half-life up to 370 min. Here we present novel X-ray crystallographic data on these mutants that provide a plausible model for the observed enhanced stability. We also more extensively characterize the previously reported variants and report on a new stabilizing mutant, L169K. The improved stability of these engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans.

  6. 12-((5-Iodo-4-azido-2-hydroxybenzoyl)amino)dodecanoic acid: Biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

    SciTech Connect

    Kinnunen, P.M.; Klopf, F.H.; Bastiani, C.A.; Gelfman, C.M.; Lange, L.G. )

    1990-02-13

    Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-((5-iodo-4-azido-2-hydroxybenzoyl)amino)dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA;cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl ({sup 14}C)oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent K{sub i} of 150 {mu}M. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions, and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of {sup 125}IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.

  7. Cellular function of neuropathy target esterase in lysophosphatidylcholine action

    SciTech Connect

    Vose, Sarah C.; Fujioka, Kazutoshi; Gulevich, Alex G.; Lin, Amy Y.; Holland, Nina T.; Casida, John E.

    2008-11-01

    Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.

  8. Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification.

    PubMed

    Berlemont, Renaud; Spee, Olivier; Delsaute, Maud; Lara, Yannick; Schuldes, Jörg; Simon, Carola; Power, Pablo; Daniel, Rolf; Galleni, Moreno

    2013-01-01

    in order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 α/β hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

  9. Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene▿ †

    PubMed Central

    Li, Xin-Liang; Skory, Christopher D.; Cotta, Michael A.; Puchart, Vladimir; Biely, Peter

    2008-01-01

    Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene aes1, encoding the acetyl esterase (Aes1) of Hypocrea jecorina, was identified by amino-terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino acid residues with the first 19 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the secreted enzyme were 37,088 Da and pH 5.89, respectively. No significant homology was found between the predicated Aes1 and carbohydrate esterases of known families, but putative aes1 orthologs were found in genomes of many fungi and bacteria that produce cell wall-degrading enzymes. The aes1 transcript levels were high when the fungal cells were induced with sophorose, cellulose, oat spelt xylan, lactose, and arabinose. The recombinant Aes1 produced by H. jecorina transformed with aes1 under the cellobiohydrolase I promoter displayed properties similar to those reported for the native enzyme. The enzyme hydrolyzed acetate ester bond specifically. Using 4-nitrophenyl acetate as substrate, the activity of the recombinant enzyme was enhanced by d-xylose, d-glucose, cellobiose, d-galactose, and xylooligosaccharides but not by arabinose, mannose, or lactose. With the use of 4-nitrophenyl-β-d-xylopyranoside monoacetate as substrate in a β-xylosidase-coupled assay, Aes1 hydrolyzed positions 3 and 4 with the same efficiency while the H. jecorina acetylxylan esterase 1 exclusively deacetylated the position 2 acetyl group. Aes1 was capable of transacetylating methylxyloside in aqueous solution. The data presented demonstrate that Aes1 and other homologous microbial proteins may represent a new family of esterases for lignocellulose biodegradation. PMID:18978092

  10. Juvenile hormone esterase: biochemistry and structure

    PubMed Central

    Kamita, Shizuo G.; Hammock, Bruce D.

    2013-01-01

    Synopsis Normal insect development requires a precisely timed, precipitous drop in hemolymph juvenile hormone (JH) titer. This drop occurs through a coordinated halt in JH biosynthesis and increase in JH metabolism. In many species, JH esterase (JHE) is critical for metabolism of the resonance-stabilized methyl ester of JH. JHE metabolizes JH with a high kcat/KM ratio that results primarily from an exceptionally low KM. Here we review the biochemistry and structure of authentic and recombinant JHEs from six insect orders, and present updated diagnostic criteria that help to distinguish JHEs from other carboxylesterases. The use of a JHE-encoding gene to improve the insecticidal efficacy of biopesticides is also discussed. PMID:23543805

  11. Cholinesterase inhibitors from botanicals

    PubMed Central

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P.; Ahmed, K. K. Mueen

    2013-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  12. A key esterase required for the mineralization of quizalofop-p-ethyl by a natural consortium of Rhodococcus sp. JT-3 and Brevundimonas sp. JT-9.

    PubMed

    Zhang, Hui; Li, Mengya; Li, Jie; Wang, Guangli; Li, Feng; Xu, Dayong; Liu, Yuan; Xiong, Minghua

    2017-04-05

    A natural consortium, named L1, of Rhodococcus sp. JT-3 and Brevundimonas sp. JT-9 was obtained from quizalofop-p-ethyl (QE) polluted soil. The consortium was able to use QE as a sole carbon source for growth and degraded 100mgL(-1) of QE in 60h. Strain JT-3 initiated the catabolism of QE to quizalofop acid (QA), which was used by strain JT-9 as carbon source for growth and to simultaneously feed strain JT-3. A novel esterase EstS-JT, which was responsible for the transformation of QE to QA and essential for the mineralization of QE by the consortium, was cloned from strain JT-3. EstS-JT showed low amino acid identity to other reported esterases from esterase family VIII and represents a new member of this family. The deduced amino acid sequence contained the esterase family VIII conserved motifs S-X-X-K, YSV and WAG. The purified recombinant EstS-JT displayed maximal esterase activity at 35°C and pH 7.5. An inhibitor assay, site-directed mutagenesis and 3D modeling analysis revealed that S64, K67 and Y175 were essential for catalysis and probably comprised the catalytic center of EstS-JT. Additionally, EstS-JT had broad substrate specificity and was capable of hydrolyzing p-nitrophenyl esters (C2-C8) and various AOPP herbicides.

  13. Characterization of a Feruloyl Esterase from Lactobacillus plantarum

    PubMed Central

    Esteban-Torres, María; Reverón, Inés; Mancheño, José Miguel; de las Rivas, Blanca

    2013-01-01

    Lactobacillus plantarum is frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant. L. plantarum WCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methyl p-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methyl p-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present among L. plantarum strains. The present study constitutes the description of feruloyl esterase activity in L. plantarum and provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species. PMID:23793626

  14. Kinetics and Molecular Docking Study of an Anti-diabetic Drug Glimepiride as Acetylcholinesterase Inhibitor: Implication for Alzheimer's Disease-Diabetes Dual Therapy.

    PubMed

    Rizvi, Syed Mohd Danish; Shaikh, Sibhghatulla; Naaz, Deeba; Shakil, Shazi; Ahmad, Adnan; Haneef, Mohd; Abuzenadah, Adel M

    2016-06-01

    At the present time, treatment of two most common degenerative disorders of elderly population i.e., Type 2 Diabetes Mellitus (T2DM) and Alzheimer's disease (AD) is a major concern worldwide. As there are several evidences that proved strong linkages between these two disorders, the idea of using dual therapeutic agent for both the diseases might be considered as a good initiative. Earlier reports have revealed that oral anti-diabetic drugs such as peroxisome proliferator activated receptor γ (PPARγ) agonists (thiazolidinediones) when used in T2DM patients suffering from AD showed improved memory and cognition. However, the underlying mechanism still needs to be deciphered. Therefore, the present study was carried out to find whether glimepiride, an oral antidiabetic drug which is a PPARγ agonist could inhibit the activity of acetylcholine esterase (AChE) enzyme. Actually, AChE inhibitors seize the breakdown of acetylcholine which forms the main therapeutic strategy for AD. Here, glimepiride showed dose dependent inhibitory activity against AChE enzyme with IC50 value of 235 μM. Kinetic analysis showed competitive inhibition, which was verified by in silico docking studies. Glimepiride was found to interact with AChE enzyme at the same locus as that of substrate acetylcholine iodide (AChI). Interestingly, amino acid residues, Q71, Y72, V73, D74, W86, N87, Y124, S125, W286, F295, F297, Y337, F338 and Y341 of AChE were found to be common for 'glimepiride-AChE interaction' as well as 'AChI-AChE interaction'. Thus the present computational and kinetics study concludes that glimepiride and other thiazolidinediones derivatives could form the basis of future dual therapy against diabetes associated neurological disorders.

  15. Nicotinic Acetylcholine Receptors in Sensory Cortex

    ERIC Educational Resources Information Center

    Metherate, Raju

    2004-01-01

    Acetylcholine release in sensory neocortex contributes to higher-order sensory function, in part by activating nicotinic acetylcholine receptors (nAChRs). Molecular studies have revealed a bewildering array of nAChR subtypes and cellular actions; however, there is some consensus emerging about the major nAChR subtypes and their functions in…

  16. Experimental measurements for the effect of dilution procedure in blood esterases as animals biomarker for exposure to OP compounds.

    PubMed

    Abass, Kasim Sakran

    2014-01-01

    Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (k(cat)/K(m)), and the K m and k cat values were 176 μM and 16,765 s(-1), respectively, for S-phenyl thioacetate ester, while detected in dilution 1: 15 was the lowest specificity constant (k(cat)/K(m)), and the Km and k cat values were 943 μM and 1154 s(-1), respectively, for acetylthiocholine iodide ester.

  17. Cell-bound lipase and esterase of Brevibacterium linens.

    PubMed

    Sorhaug, T; Ordal, Z J

    1974-03-01

    The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity.

  18. The conformation of acetylcholine at its target site in the membrane-embedded nicotinic acetylcholine receptor

    PubMed Central

    Williamson, P. T. F.; Verhoeven, A.; Miller, K. W.; Meier, B. H.; Watts, A.

    2007-01-01

    The conformation of the neurotransmitter acetylcholine bound to the fully functional nicotinic acetylcholine receptor embedded in its native membrane environment has been characterized by using frequency-selective recoupling solid-state NMR. Six dipolar couplings among five resolved 13C-labeled atoms of acetylcholine were measured. Bound acetylcholine adopts a bent conformation characterized with a quaternary ammonium-to-carbonyl distance of 5.1 Å. In this conformation, and with its orientation constrained to that previously determined by us, the acetylcholine could be docked satisfactorily in the agonist pocket of the agonist-bound, but not the agonist-free, crystal structure of a soluble acetylcholine-binding protein from Lymnaea stagnali. The quaternary ammonium group of the acetylcholine was determined to be within 3.9 Å of five aromatic residues and its acetyl group close to residues C187/188 of the principle and residue L112 of the complementary subunit. The observed >CO chemical shift is consistent with H bonding to the nicotinic acetylcholine receptor residues γY116 and δT119 that are homologous to L112 in the soluble acetylcholine-binding protein. PMID:17989232

  19. Allosteric binding sites on muscarinic acetylcholine receptors.

    PubMed

    Wess, Jürgen

    2005-12-01

    In this issue of Molecular Pharmacology, Tränkle et al. (p. 1597) present new findings regarding the existence of a second allosteric site on the M2 muscarinic acetylcholine receptor (M2 mAChR). The M2 mAChR is a prototypic class A G protein-coupled receptor (GPCR) that has proven to be a very useful model system to study the molecular mechanisms involved in the binding of allosteric GPCR ligands. Previous studies have identified several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine and the bis-pyridinium derivative 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3), which, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors >1. By analyzing the interactions of tacrine and Duo3 with other allosteric muscarinic agents predicted to bind to the previously identified ;common' allosteric binding site, Tränkle et al. provide evidence suggesting that two allosteric agents and one orthosteric ligand may be able to bind to the M2 mAChR simultaneously. Moreover, studies with mutant mAChRs indicated that the M2 receptor epitopes involved in the binding of tacrine and Duo3 may not be identical. Molecular modeling and ligand docking studies suggested that the additional allosteric site probably represents a subdomain of the receptor's allosteric binding cleft. Because allosteric binding sites have been found on many other GPCRs and drugs interacting with these sites are thought to have great therapeutic potential, the study by Tränkle et al. should be of considerable general interest.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic d...

  1. High-throughput screening method for lipases/esterases.

    PubMed

    Mateos-Díaz, Eduardo; Rodríguez, Jorge Alberto; de Los Ángeles Camacho-Ruiz, María; Mateos-Díaz, Juan Carlos

    2012-01-01

    High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.

  2. Acetylcholine Esterase Activity and Behavioral Response in Hypoxia Induced Neonatal Rats: Effect of Glucose, Oxygen and Epinephrine Supplementation

    ERIC Educational Resources Information Center

    Chathu, Finla; Krishnakumar, Amee; Paulose, Cheramadathikudyil S.

    2008-01-01

    Brain damage due to an episode of hypoxia remains a major problem in infants causing deficit in motor and sensory function. Hypoxia leads to neuronal functional failure, cerebral palsy and neuro-developmental delay with characteristic biochemical and molecular alterations resulting in permanent or transitory neurological sequelae or even death.…

  3. A New Family of Carbohydrate Esterases Is Represented by a GDSL Hydrolase/Acetylxylan Esterase from Geobacillus stearothermophilus*

    PubMed Central

    Alalouf, Onit; Balazs, Yael; Volkinshtein, Margarita; Grimpel, Yael; Shoham, Gil; Shoham, Yuval

    2011-01-01

    Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in the CAZy Database. The axe2 gene is induced by xylose, and the purified gene product completely deacetylates xylobiose peracetate (fully acetylated) and hydrolyzes the synthetic substrates 2-naphthyl acetate, 4-nitrophenyl acetate, 4-methylumbelliferyl acetate, and phenyl acetate. The pH profiles for kcat and kcat/Km suggest the existence of two ionizable groups affecting the binding of the substrate to the enzyme. Using NMR spectroscopy, the regioselectivity of Axe2 was directly determined with the aid of one-dimensional selective total correlation spectroscopy. Methyl 2,3,4-tri-O-acetyl-β-d-xylopyranoside was rapidly deacetylated at position 2 or at positions 3 and 4 to give either diacetyl or monoacetyl intermediates, respectively; methyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside was initially deacetylated at position 6. In both cases, the complete hydrolysis of the intermediates occurred at a much slower rate, suggesting that the preferred substrate is the peracetate sugar form. Site-directed mutagenesis of Ser-15, His-194, and Asp-191 resulted in complete inactivation of the enzyme, consistent with their role as the catalytic triad. Overall, our results show that Axe2 is a serine acetylxylan esterase representing a new carbohydrate esterase family. PMID:21994937

  4. Subtype-selective nicotinic acetylcholine receptor agonists enhance the responsiveness to citalopram and reboxetine in the mouse forced swim test.

    PubMed

    Andreasen, Jesper T; Nielsen, Elsebet Ø; Christensen, Jeppe K; Olsen, Gunnar M; Peters, Dan; Mirza, Naheed R; Redrobe, John P

    2011-10-01

    Nicotine increases serotonergic and noradrenergic neuronal activity and facilitates serotonin and noradrenaline release. Accordingly, nicotine enhances antidepressant-like actions of reuptake inhibitors selective for serotonin or noradrenaline in the mouse forced swim test and the mouse tail suspension test. Both high-affinity α4β2 and low-affinity α7 nicotinic acetylcholine receptor subtypes are implicated in nicotine-mediated release of serotonin and noradrenaline. The present study therefore investigated whether selective agonism of α4β2 or α7 nicotinic acetylcholine receptors would affect the mouse forced swim test activity of two antidepressants with distinct mechanisms of action, namely the selective serotonin reuptake inhibitor citalopram and the noradrenaline reuptake inhibitor reboxetine. Subthreshold and threshold doses of citalopram (3 and 10 mg/kg) or reboxetine (10 and 20 mg/kg) were tested alone and in combination with the novel α4β2-selective partial nicotinic acetylcholine receptor agonist, NS3956 (0.3 and 1.0 mg/kg) or the α7-selective nicotinic acetylcholine receptor agonist, PNU-282987 (10 and 30 mg/kg). Alone, NS3956 and PNU-282987 were devoid of activity in the mouse forced swim test, but both 1.0 mg/kg NS3956 and 30 mg/kg PNU-282987 enhanced the effect of citalopram and also reboxetine. The data suggest that the activity of citalopram and reboxetine in the mouse forced swim test can be enhanced by agonists at either α4β2 or α7 nicotinic acetylcholine receptors, suggesting that both nicotinic acetylcholine receptor subtypes may be involved in the nicotine-enhanced action of antidepressants.

  5. 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.

    PubMed

    Ramya, Shanivarsanthe Leelesh; Venkatesan, Thiruvengadam; Srinivasa Murthy, Kottilingam; Jalali, Sushil Kumar; Verghese, Abraham

    2016-01-01

    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 2.32μmol/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 0.26μmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

  6. Metabolism of acetylcholine in human erythrocytes

    SciTech Connect

    Chapman, E.S.

    1990-01-01

    In order to examine the possible role of erythrocyte acetylcholinesterase in the maintenance of membrane phospholipid content and membrane fluidity, experiments were performed to monitor the activity of the enzyme and follow the fate of one of its hydrolytic products, choline. Intact human erythrocytes were incubated with acetylcholine (choline methyl-{sup 14}C). The incubation resulted in the hydrolysis of acetylcholine to acetate and choline; the reaction was catalyzed by membrane acetylcholinesterase. The studies demonstrate the further metabolism of choline. Experiments were carried out to determine rate of hydrolysis of acetylcholine, uptake of choline, identification of intracellular metabolites of choline, and identification of radiolabeled membrane components. Erythrocytes at a 25% hematocrit were incubated in an isoosmotic bicarbonate buffer pH 7.4, containing glucose, adenosine, streptomycin and penicillin with 0.3 {mu}Ci of acetylcholine (choline methyl-{sup 14}C), for 24 hours. Aliquots of the erythrocyte suspension were taken throughout for analysis. Erythrocytes were washed free of excess substrate, lysed, and the hemolysate was extracted for choline and its metabolites. Blank samples containing incubation buffer and radiolabeled acetylcholine only, and erythrocyte hemolysate extracts were analyzed for choline content, the difference between blank samples and hemolysate extracts was the amount of choline originating from acetylcholine and attributable to acetylcholinesterase activity. The conversion of choline to {sup 14}C-betaine is noted after several minutes of incubation; at 30 minutes, more than 80% of {sup 14}C-choline is taken up and after several hours, detectable levels of radiolabeled S-adenosylmethionine were present in the hemolysate extract.

  7. A New Functional Classification of Glucuronoyl Esterases by Peptide Pattern Recognition

    PubMed Central

    Agger, Jane W.; Busk, Peter K.; Pilgaard, Bo; Meyer, Anne S.; Lange, Lene

    2017-01-01

    Glucuronoyl esterases are a novel type of enzymes believed to catalyze the hydrolysis of ester linkages between lignin and glucuronoxylan in lignocellulosic biomass, linkages known as lignin carbohydrate complexes. These complexes contribute to the recalcitrance of lignocellulose. Glucuronoyl esterases are a part of the microbial machinery for lignocellulose degradation and coupling their role to the occurrence of lignin carbohydrate complexes in biomass is a desired research goal. Glucuronoyl esterases have been assigned to CAZymes family 15 of carbohydrate esterases, but only few examples of characterized enzymes exist and the exact activity is still uncertain. Here peptide pattern recognition is used as a bioinformatic tool to identify and group new CE15 proteins that are likely to have glucuronoyl esterase activity. 1024 CE15-like sequences were drawn from GenBank and grouped into 24 groups. Phylogenetic analysis of these groups made it possible to pinpoint groups of putative fungal and bacterial glucuronoyl esterases and their sequence variation. Moreover, a number of groups included previously undescribed CE15-like sequences that are distinct from the glucuronoyl esterases and may possibly have different esterase activity. Hence, the CE15 family is likely to comprise other enzyme functions than glucuronoyl esterase alone. Gene annotation in a variety of fungal and bacterial microorganisms showed that coprophilic fungi are rich and diverse sources of CE15 proteins. Combined with the lifestyle and habitat of coprophilic fungi, they are predicted to be excellent candidates for finding new glucuronoyl esterase genes. PMID:28293230

  8. Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester.

    PubMed

    Julien-David, Diane; Ennahar, Saïd; Miesch, Michel; Geoffroy, Philippe; Raul, Francis; Aoude-Werner, Dalal; Lessinger, Jean-Marc; Marchioni, Eric

    2009-10-01

    Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.

  9. Overexpression of esterase D in kidney from trisomy 13 fetuses.

    PubMed Central

    Loughna, S; Bennett, P; Gau, G; Nicolaides, K; Blunt, S; Moore, G

    1993-01-01

    Human trisomy 13 (Patau syndrome) occurs in approximately 1 in 5,000 live births. It is compatible with life, but prolonged survival is rare. Anomalies often involve the urogenital, cardiac, craniofacial, and central nervous systems. It is possible that these abnormalities may be due to the overexpression of developmentally important genes on chromosome 13. The expression of esterase D (localized to chromosome 13q14.11) has been investigated in both muscle and kidney from trisomy 13 fetuses and has been compared with normal age- and sex-matched fetal tissues, by using northern analysis. More than a twofold increase in expression of esterase D was found in the kidney of two trisomy 13 fetuses, with normal levels in a third. Overexpression was not seen in the muscle tissues from these fetuses. Images Figure 1 Figure 2 Figure 3 PMID:8213811

  10. [On the problem of histochemical demonstration of esterase activity by the thiolacetic acid method in the acellular slime mold Physarum confertum (author's translation)].

    PubMed

    Dierkes, U

    1977-01-01

    The application of the histochemical thiolacetic acid method on plasmodia of the acellular slime mold Physarum confertum leads to the formation of lead sulfide deposits at the outer cytoplasmic surface and its invaginations. The reaction cannot be reduced by esterase- and cholin/acetylcholinesterase inhibitors. Successive application of lead and sulfide in the absence of substrate results in a lead sulfide deposit at the same sites indicating that the underlying reaction is based on an artificial adsorption of ions at the surface of the plasmodium. This finding means that the thiolacetic acid method is not suited for the demonstration of a surface-associated esterase/cholinesterase activity in slime molds. Based on the ion adsorption property of the surface of plasmodia a simple method is developed for the "in toto" demonstration of the plasmamembrane-invagination-system in aceullar slime molds.

  11. Experimental Measurements for the Effect of Dilution Procedure in Blood Esterases as Animals Biomarker for Exposure to OP Compounds

    PubMed Central

    Abass, Kasim Sakran

    2014-01-01

    Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (kcat/Km), and the Km and kcat values were 176 μM and 16,765 s−1, respectively, for S-phenyl thioacetate ester, while detected in dilution 1 : 15 was the lowest specificity constant (kcat/Km), and the Km and kcat values were 943 μM and 1154 s−1, respectively, for acetylthiocholine iodide ester. PMID:24864243

  12. [Phosphonate esterase activity in human serum (author's transl)].

    PubMed

    Labadie, M; Laplaud, P M; Lachatre, G; Breton, J C

    1980-02-01

    A simple methodology for the spectrophotometric assay of phosphonate esterase activity in human serum samples is described, featuring incubation at 30 degrees C in a medium containing p-nitrophenol and phenyl-phosphonic acid ester. Reproducibility of the method as well a mean values in normal patients vs age and sex are reported. Serum activity appears to be increased almost exclusively during pregnancy or administration of estrogenic drugs (as oral contraceptives or in prostate neoplasms).

  13. Nicotinic acetylcholine receptor from chick optic lobe.

    PubMed Central

    Norman, R I; Mehraban, F; Barnard, E A; Dolly, J O

    1982-01-01

    An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors. Images PMID:6175967

  14. Primary Structure of Nicotinic Acetylcholine Receptor

    DTIC Science & Technology

    1986-08-01

    quantities of starting material (for reviews of receptor, see Popot and Changeux, 1984; Stroud and Finer-Moore, 1985). This work led to the...Cloning of the Acetylcholine Receptor. Cold Spring Harbor Symp. on Quant. Biol. XLVIH: 71-78. 15. Popot , J-L. and Changeux, J-P. (1984) The

  15. Turnover of Acetylcholine Receptors: Mechanisms of Regulation

    DTIC Science & Technology

    1988-12-01

    ME, Whittingham S, and Duane DD (1976) Antibody to acetylcholine receptor in myasthenia gravis : prevalance, clinical correlates and diagnostic value...transferred to nitorcellulose. Proc Natl Acad Sci 77:5201-5205. Weinberg CB and Hall ZW (1979) Antibodies from patients with myasthenia gravis recognize

  16. Characterisation of a New Family of Carboxyl Esterases with an OsmC Domain

    PubMed Central

    Horsfall, Louise E.; Wardrope, Caroline; Togneri, Peter D.; Marles-Wright, Jon; Rosser, Susan J.

    2016-01-01

    Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended ‘lid’ region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries. PMID:27851780

  17. Isozymic variations in specific and nonspecific esterase and its thermostability in silkworm, Bombyx mori L.

    PubMed

    Patnaik, Bharat Bhusan; Biswas, Tapati Datta; Nayak, Sandeepta Kumar; Saha, A K; Majumdar, M K

    2012-09-01

    Esterase isozymic variations were documented in the haemolymph of developed multivoltine and bivoltine silkworm breeds during unfavorable seed crop seasons of May - September using á- and â- napthylacetate separately to identify specific and nonspecific esterase having thermotolerant potentiality. Variations existed in the isozyme pattern with three bands (Est-2, 3 and 4) in pure Nistari race and other developed multivoltine and bivoltine breeds. Est-2 and Est-3 were non-specific esterases as they were observed when both á- and â-napthylacetate was used as substrates separately. Est-4 band was observed only with á-napthylacetate as substrate and was therefore confirmed to be specific á-esterase band in the haemolymph of silkworm, Bombyx mori L. Zymograms showed that the non-specific esterase band (Est-3) with R1 of 0.43 and specific á-esterase band (Est-4) with R(f) of 0.32 predominately withstood a temperature of 70 +/- 2 degrees C for a duration of 10 min and were confirmed as thermostable esterases in haemolymph of silkworm, Bombyx mori L. This also categorized the presence of thermostable esterases in developed multivoltine and bivoltine breeds of silkworm, even though the qualitative activity was more in the former than the latter. The qualitative presence of thermostable esterases and their activity could be adopted as an indicative biochemical marker in relation to thermotolerance in silkworm.

  18. The effect of liver esterases and temperature on remifentanil degradation in vitro.

    PubMed

    Piazza, Ornella; Cascone, Sara; Sessa, Linda; De Robertis, Edoardo; Lamberti, Gaetano

    2016-08-20

    Remifentanil is a potent opioid metabolized by serum and tissue esterases; it is routinely administered to patients with liver failure as anaesthetic and analgo-sedative without variation in doses, even if prolonged clinical effects and respiratory depression have been observed in these patients. The aim of this study was to determine remifentanil enzymatic degradation kinetics bearing in mind the effect of liver esterases in order to trace a more accurate pharmacokinetic profile of the drug. Solution samples were taken over time and analysed to measure remifentanil concentration by HPLC. We reproduced the physiological settings, varying temperature and pH in vitro and evaluated the kinetics of degradation of remifentanil in the presence of Rhizopus Oryzae esterases, equine liver esterases and porcine liver esterases. Remifentanil kinetics of degradation was accelerated by porcine liver esterases. Remifentanil in vitro half-life decreases with increasing temperatures in the presence of porcine liver esterases. A drug model simulation considering the effect of temperature in the presence of liver esterases was developed. Remifentanil in vitro half-life decreases with increasing temperatures when porcine liver esterases are present. In this paper we propose a model for describing remifentanil degradation kinetics at various temperatures.

  19. Effects of acute chlorpyrifos exposure on in vivo acetylcholine accumulation in rat striatum

    SciTech Connect

    Karanth, Subramanya; Liu, Jing; Mirajkar, Nikita; Pope, Carey . E-mail: carey.pope@okstate.edu

    2006-10-01

    This study examined the acute effects of chlorpyrifos (CPF) on cholinesterase inhibition and acetylcholine levels in the striatum of freely moving rats using in vivo microdialysis. Adult, male Sprague-Dawley rats were treated with vehicle (peanut oil, 2 ml/kg) or CPF (84, 156 or 279 mg/kg, sc) and functional signs of toxicity, body weight and motor activity recorded. Microdialysis was conducted at 1, 4 and 7 days after CPF exposure for measurement of acetylcholine levels in striatum. Rats were then sacrificed and the contralateral striatum and diaphragm were collected for biochemical measurements. Few overt signs of cholinergic toxicity were noted in any rats. Body weight gain was significantly affected in the high-dose (279 mg/kg) group only, while motor activity (nocturnal rearing) was significantly reduced in all CPF-treated groups at one day (84 mg/kg) or from 1-4 days (156 and 279 mg/kg) after dosing. Cholinesterase activities in both diaphragm and striatum were markedly inhibited (50-92%) in a time-dependent manner, but there were relatively minimal dose-related changes. In contrast, time- and dose-dependent changes in striatal acetylcholine levels were noted, with significantly higher levels noted in the high-dose group compared to other groups. Maximal increases in striatal acetylcholine levels were observed at 4-7 days after dosing (84 mg/kg, 7-9-fold; 156 mg/kg, 10-13-fold; 279 mg/kg, 35-57-fold). Substantially higher acetylcholine levels were noted when an exogenous cholinesterase inhibitor was included in the perfusion buffer, but CPF treatment-related differences were substantially lower in magnitude under those conditions. The results suggest that marked differences in acetylcholine accumulation can occur with dosages of CPF eliciting relatively similar degrees of cholinesterase inhibition. Furthermore, the minimal expression of classic signs of cholinergic toxicity in the presence of extensive brain acetylcholine accumulation suggests that some

  20. Constructs of human neuropathy target esterase catalytic domain containing mutations related to motor neuron disease have altered enzymatic properties.

    PubMed

    Hein, Nichole D; Stuckey, Jeanne A; Rainier, Shirley R; Fink, John K; Richardson, Rudy J

    2010-07-01

    Neuropathy target esterase (NTE) is a phospholipase/lysophospholipase associated with organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). Distal degeneration of motor axons occurs in both OPIDN and the hereditary spastic paraplegias (HSPs). Recently, mutations within the esterase domain of NTE were identified in patients with a novel type of HSP (SPG39) designated NTE-related motor neuron disease (NTE-MND). Two of these mutations, arginine 890 to histidine (R890H) and methionine 1012 to valine (M1012V), were created in human recombinant NTE catalytic domain (NEST) to measure possible changes in catalytic properties. These mutated enzymes had decreased specific activities for hydrolysis of the artificial substrate, phenyl valerate. In addition, the M1012V mutant exhibited a reduced bimolecular rate constant of inhibition (k(i)) for all three inhibitors tested: mipafox, diisopropylphosphorofluoridate, and chlorpyrifos oxon. Finally, while both mutated enzymes inhibited by OP compounds exhibited altered time-dependent loss of their ability to be reactivated by nucleophiles (aging), more pronounced effects were seen with the M1012V mutant. Taken together, the results from specific activity, inhibition, and aging experiments suggest that the mutations found in association with NTE-MND have functional correlates in altered enzymological properties of NTE.

  1. Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location.

    PubMed

    Hamers, Timo; van den Brink, Paul J; Mos, Lizzy; van der Linden, Sander C; Legler, Juliette; Koeman, Jan H; Murk, Albertinka J

    2003-01-01

    In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between and were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in than in and . Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for aquatic organisms and was highest in . Although estrogenic potency of rainwater correlated with OC concentrations, the ER-CALUX responses could not be attributed to

  2. Agonists block currents through acetylcholine receptor channels.

    PubMed Central

    Sine, S M; Steinbach, J H

    1984-01-01

    We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR. PMID:6478036

  3. Neuronal Nicotinic Acetylcholine Receptors and Epilepsy

    PubMed Central

    Bertrand, Daniel

    2002-01-01

    The identification of a genetically transmissible form of epilepsy that is associated with a mutation in CHRNA4, the gene that encodes the α4 subunit of the high-affinity nicotinic acetylcholine receptor, was the first demonstration that an alteration in a ligand-gated ion channel can cause seizures. Since then, nine mutations have been found, and analysis of their physiologic properties has revealed that all of them enhance receptor function. PMID:15309115

  4. External Imaging of Cerebral Muscarinic Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

    Eckelman, William C.; Reba, Richard C.; Rzeszotarski, Waclaw J.; Gibson, Raymond E.; Hill, Thomas; Holman, B. Leonard; Budinger, Thomas; Conklin, James J.; Eng, Robert; Grissom, Michael P.

    1984-01-01

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  5. External imaging of cerebral muscarinic acetylcholine receptors

    SciTech Connect

    Eckelman, W.C.; Reba, R.C.; Rzeszotarski, W.J.; Gibson, R.E.; Hill, T.; Holman, B.L.; Budinger, T.; Conklin, J.J.; Eng, R.; Grissom, M.P.

    1984-01-20

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  6. Esterase activity of BSA-ZnO nanoparticle complex

    NASA Astrophysics Data System (ADS)

    Bhogale, A.; Nair, A.; Patel, N.; Miotello, A.; Kothari, D. C.

    2014-04-01

    The effect of Zinc Oxide Nanoparticles (ZnO NPs) on functional properties of Bovine Serum Albumin (BSA) protein was studied. ZnO NPs were synthesized with average size of ˜7.5 nm as obtained from TEM analysis. The catalytic conversion of p-nitrophenylacetate (PNPA) to p-nitrophenol in the presence of BSA attached with ZnO NPs was examined by UV-Vis spectroscopy at room temperature. The result suggests that esterase activity of BSA is significantly enhanced (6 times) due to the ground state BSA-ZnO complex formation.

  7. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

    DOEpatents

    Arnold, F.H.; Moore, J.C.

    1998-04-21

    A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases. These enzymes exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

  8. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

    DOEpatents

    Arnold, Frances H.; Moore, Jeffrey C.

    1998-01-01

    A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

  9. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  10. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

    DOEpatents

    Arnold, Frances H.; Moore, Jeffrey C.

    1999-01-01

    A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

  11. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  12. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme... animals. (c) The enzyme is produced by a process which completely removes the organism Mucor miehei...

  13. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  14. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  15. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

    DOEpatents

    Arnold, F.H.; Moore, J.C.

    1999-05-25

    A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

  16. A Comparison of Multiple Esterases as Biomarkers of Organophosphate Exposure and Effect in Two Earthworm Species

    PubMed Central

    Schneider, Ashley; Stoskopf, Michael K.

    2011-01-01

    Two different earthworm species, Eisenia fetida and Lumbricus terrestris, were exposed to 5 μg/cm2 of malathion to evaluate their usefulness as sentinels of organophosphate exposure and to assess three different esterases, as biomarkers of malathion exposure and effect. Tissue xenobiotic burdens and esterase activity were determined for each species and each esterase in order to assess variability. E. fetida exhibited 4-fold less variability in tissue burdens than did L. terrestris and had less variable basal esterase activities. An attempt was made to correlate malathion and malaoxon tissue burdens with esterase activity post-exposure. There was no malaoxon present in the earthworm tissues. No significant correlations were determined by comparing acetylcholinesterase, butyrylcholinesterase, nor carboxylesterase activities with malathion burdens. PMID:21404045

  17. [Sites of synthesis of acetylcholine receptors in denervated muscles].

    PubMed

    Giacobini Robecchi, M G; Garelli, M; Filogamo, G

    1980-09-01

    Muscle fibres binding with 125I alpha-bungarotoxine from Bungarus Multicinctus, after treatment with saponine, shows (in electron microscope autoradiography) intracellular binding sites identifying sites of acetylcholine receptor synthesis. In innervated muscle, the acetylcholine receptor is located only at the neuromuscular junction. In denervated muscle the receptor is distributed along the whole sarcolemma; in this situation the acetylcholine receptor is synthesized "ex novo" in the membrane system over the whole length of the muscle fibre.

  18. Immunocytochemical Detection of Acetylcholine in the Rat Central Nervous System

    NASA Astrophysics Data System (ADS)

    Geffard, M.; McRae-Degueurce, A.; Souan, Marie Laure

    1985-07-01

    A specific antibody to acetylcholine was raised and used as a marker for cholinergic neurons in the rat central nervous system. The acetylcholine conjugate was obtained by a two-step immunogen synthesis procedure. An enzyme-linked immunosorbent assay was used to test the specificity and affinity of the antibody in vitro; the results indicated high affinity. A chemical perfusion mixture of allyl alcohol and glutaraldehyde was used to fix the acetylcholine in the nervous tissue. Peroxidase-antiperoxidase immunocytochemistry showed many acetylcholine-immunoreactive cells and fibers in sections from the medial septum region.

  19. Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus.

    PubMed

    Wang, Mingyang; Veit, Michael

    2016-01-01

    Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.

  20. Vine Trimming Shoots as Substrate for Ferulic Acid Esterases Production.

    PubMed

    Pérez-Rodríguez, N; Outeiriño, D; Torrado Agrasar, A; Domínguez, J M

    2017-02-01

    Ferulic acid esterases (FAE) possess a large variety of biotechnological applications mainly based on their ability to release ferulic acid from lignocellulosic matrixes. The use of vine trimming shoots (VTS), an agricultural waste, as substrate for the generation of this kind of esterases represents an attractive alternative to change the consideration of VTS from residue to resource. Furthermore, xylanase, cellobiase, and cellulase activities were quantified. Six microorganisms were screened for FAE production by solid-state fermentation, and the effects of the additional supplementation and substrate size were also tested. Finally, the process was scaled-up to a horizontal bioreactor where the influence of aeration in enzymatic activities was evaluated. Thus, the optimal FAE activity (0.44 U/g dry VTS) was attained by Aspergillus terreus CECT 2808, in non-additional supplementation media, using the larger particles size of substrate (≤ 5 mm) and at a flow rate of 0.7 L/min.

  1. A Mass Spectrometry “Sensor” for in Vivo Acetylcholine Monitoring

    PubMed Central

    Song, Peng; Hershey, Neil D.; Mabrouk, Omar S.; Slaney, Thomas R.; Kennedy, Robert T.

    2012-01-01

    Developing sensors for in vivo chemical monitoring is a daunting challenge. An alternative approach is to couple sampling methods with online analytical techniques; however, such approaches are generally hampered by lower temporal resolution and slow analysis. In this work, microdialysis sampling was coupled with segmented flow electrospray ionization mass spectrometry (ESI-MS) to perform in vivo chemical monitoring. Use of segmented flow to prevent Taylor dispersion of collected zones and rapid analysis with direct ESI-MS allowed 5 s temporal resolution to be achieved. The MS “sensor” was applied to monitoring acetylcholine in the brain of live rats. The detection limit of 5 nM was sufficient to monitor basal acetylcholine as well as dynamic changes elicited by microinjection of neostigmine, an inhibitor of acetycholinesterase that evoked rapid increases in acetycholine, and tetrodotoxin, a blocker of Na+ channels, that lowered the acetylcholine concentration. The versatility of the sensor was demonstrated by simultaneously monitoring metabolites and infused drugs. PMID:22616788

  2. Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine

    PubMed Central

    1980-01-01

    The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen. PMID:7391135

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

    PubMed

    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.

  4. Relative potencies of the four stereoisomers of isomalathion for inhibition of hen brain acetylcholinesterase and neurotoxic esterase in vitro.

    PubMed

    Jianmongkol, S; Berkman, C E; Thompson, C M; Richardson, R J

    1996-08-01

    The cholinergic toxicity of malathion is exacerbated by its isomerization product, isomalathion, which inhibits detoxifying carboxylesterases as well as target acetylcholinesterase (AChE). Previous work has shown that the four stereoisomers of isomalathion, (1R, 3R), (1R, 3S), (1S, 3R), and (1S, 3S), differ in their inhibitory potencies against either rat brain or electric eel AChE. The present study examined the relative inhibitory potencies of these stereoisomers and the totally racemic mixture (1RS, 3RS) against hen brain AChE and neurotoxic esterase (NTE) to provide new data on stereoselective inhibition of neurotoxicologically significant esterases and to assess the potential of these compounds to cause organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). The order of potencies against hen brain AChE was (1R, 3R) > (1R, 3S) > (1RS, 3RS) > (1S, 3R) > (1S, 3S), with a 15-fold difference between the strongest (ki = 388 mM-1 min-1; 20 min I50 = 89.3 nM) and weakest (ki = 25.6 mM-1 min-1; 20 min I50 = 1354 nM) inhibitors. Both asymmetric centers contributed substantially and interdependently to inhibitory potency, but the effect of changing the configuration at phosphorus alone was greater than changing the configuration at carbon alone. None of the isomalathions was an effective inhibitor of hen brain NTE (extrapolated 20 min I50 values were 1.2 to 29 mM), yielding NTE/ AChE I50 ratios (neuropathy target ratios, NTRs) of 1.5 x 10(3) to 1.5 x 10(5). NTRs of this magnitude indicate that none of the isomalathions should initiate OPIDN, even after doses greatly exceeding the LD50. Therefore, reports of OPIDN or other neuropathic sequelae associated with malathion exposures in humans cannot be explained on the basis of NTE inhibition by contaminating isomalathions.

  5. Oseltamivir blocks human neuronal nicotinic acetylcholine receptor-mediated currents.

    PubMed

    Muraki, Katsuhiko; Hatano, Noriyuki; Suzuki, Hiroka; Muraki, Yukiko; Iwajima, Yui; Maeda, Yasuhiro; Ono, Hideki

    2015-02-01

    The effects of oseltamivir, a neuraminidase inhibitor, were tested on the function of neuronal nicotinic acetylcholine receptors (nAChRs) in a neuroblastoma cell line IMR32 derived from human peripheral neurons and on recombinant human α3β4 nAChRs expressed in HEK cells. IMR32 cells predominately express α3β4 nAChRs. Nicotine (nic, 30 μm)-evoked currents recorded at -90 mV in IMR32 cells using the whole-cell patch clamp technique were reversibly blocked by oseltamivir in a concentration-dependent manner. In contrast, an active metabolite of oseltamivir, oseltamivir carboxylate (OC) at 30 μm had little effect on the nic-evoked currents. Oseltamivir also blocked nic-evoked currents derived from HEK cells with recombinant α3β4 nAChRs. This blockade was voltage-dependent with 10, 30 and 100 μm oseltamivir inhibiting ~50% at -100, -60 and -40 mV, respectively. Non-inactivating currents in IMR32 cells and in HEK cells with α3β4 nAChRs, which were evoked by an endogenous nicotinic agonist, ACh (5 μm), were reversibly blocked by oseltamivir. These data demonstrate that oseltamivir blocks nAChRs, presumably via binding to a site in the channel pore.

  6. Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

    PubMed

    Ockenga, Wymke; Tikkanen, Ritva

    2015-05-12

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

  7. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Tikkanen, Ritva

    2015-01-01

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles. PMID:25985102

  8. Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences.

    PubMed

    Aurilia, V; Martin, J C; McCrae, S I; Scott, K P; Rincon, M T; Flint, H J

    2000-06-01

    Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain. A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain. The esterase domains from CesA and XynB share significant sequence identity (44%) and belong to carbohydrate esterase family 3; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity. The esterase domain of XynE, however, shares 42% amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex. The dockerin sequences of CesA and XynE differ significantly from those previously described in R. flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.

  9. An organic-solvent-tolerant esterase from thermophilic Bacillus licheniformis S-86.

    PubMed

    Torres, Sebastián; Martínez, M Alejandra; Pandey, Ashok; Castro, Guillermo R

    2009-01-01

    A thermophile, halotolerant and organic-solvent-tolerant esterase producer Bacillus sp. S-86 strain previously isolated was found to belong to Bacillus licheniformis species through morphological, biochemical, 16S rRNA gene sequence analyses and rDNA intergenic spacers amplification (ITS-PCR). The strain can grow at 55 degrees C in presence of C2-C7 alkanols (log P=-0.86 to 2.39), and NaCl concentrations up to 15% (w/v). This bacterium showed optimal growth and esterase production at 50 degrees C. Two different molecular weight esterase activities were detected in zymographic assays. PMSF inhibited type I esterase activity, showing no inhibitory effect on type II esterase activity. B. licheniformis S-86 was able to grow in presence of hydroxylic organic-solvents like propan-2-ol, butan-1-ol and 3-methylbutan-1-ol. At a sub-lethal concentration of these solvents (392 mmoll(-1) propan-2-ol; 99 mmol l(-1) butan-1-ol, 37 mmol l(-1) 3-methylbutan-1-ol), adequate to produce 50% cell growth inhibition at 50 degrees C, an increment between 1.9 and 2.3 times was observed in type I esterase production, and between 2.2 and 3.1 times in type II esterase production.

  10. Comparative effects of niflumic acid and nifedipine on 5-hydroxytryptamine- and acetylcholine-induced contraction of the rat trachea.

    PubMed

    Teixeira, M C; Coelho, R R; Leal-Cardoso, J H; Criddle, D N

    2000-04-07

    The effects of niflumic acid, an inhibitor of Ca(2+)-activated Cl(-) (Cl((Ca))) channels, were compared with those of the voltage-dependent Ca(2+) channel (VDCC) blocker nifedipine on 5-hydroxytryptamine (5-HT)- and acetylcholine-induced contractions of the rat isolated trachea. Niflumic acid (3-100 microM) induced a concentration-dependent inhibition of 5-HT (10 microM)-induced contractions, with a reduction to 37.0+/-9.5% of the control at the highest concentration. One micromolar nifedipine, which completely blocked 60 mM KCl-induced contractions, reduced the response to 5-HT similarly to 39.2+/-11.5% of the control. The inhibition of the 5-HT response was not significantly different from that produced by the combined presence of nifedipine (1 microM) and niflumic acid (100 microM), suggesting that their effects were not additive. In contrast, neither niflumic acid (3-100 microM) nor nifedipine (1 microM) inhibited acetylcholine-induced contractions. The contraction to 5-HT (10 microM) in Cl(-)-free solution was decreased by more than approximately 85% of the control, whilst that of acetylcholine was reduced only by approximately 36%. Our data show that niflumic acid exerts selective inhibitory effects on 5-HT-induced contraction, and suggest that activation of Cl((Ca)) channels may be a mechanism whereby 5-HT (but not acetylcholine) induces Ca(2+) entry via VDCCs to elicit contraction.

  11. Heterologous Expression of Two Ferulic Acid Esterases from Penicillium funiculosum

    NASA Astrophysics Data System (ADS)

    Knoshaug, Eric P.; Selig, Michael J.; Baker, John O.; Decker, Stephen R.; Himmel, Michael E.; Adney, William S.

    Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

  12. Heterologous Expression of Two Ferulic Acid Esterases from Penicillium Funiculosum

    SciTech Connect

    Knoshaug, E. P.; Selig, M. J.; Baker, J. O.; Decker, S. R.; Himmel, M. E.; Adney, W. S.

    2008-01-01

    Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

  13. Binding of rabies virus to purified Torpedo acetylcholine receptor.

    PubMed

    Lentz, T L; Benson, R J; Klimowicz, D; Wilson, P T; Hawrot, E

    1986-12-01

    The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

  14. Exercise and neuromodulators: choline and acetylcholine in marathon runners

    NASA Technical Reports Server (NTRS)

    Conlay, L. A.; Sabounjian, L. A.; Wurtman, R. J.

    1992-01-01

    Certain neurotransmitters (i.e., acetylcholine, catecholamines, and serotonin) are formed from dietary constituents (i.e., choline, tyrosine and tryptophan). Changing the consumption of these precursors alters release of their respective neurotransmitter products. The neurotransmitter acetylcholine is released from the neuromuscular junction and from brain. It is formed from choline, a common constituent in fish, liver, and eggs. Choline is also incorporated into cell membranes; membranes may likewise serve as an alternative choline source for acetylcholine synthesis. In trained athletes, running a 26 km marathon reduced plasma choline by approximately 40%, from 14.1 to 8.4 uM. Changes of similar magnitude have been shown to reduce acetylcholine release from the neuromuscular junction in vivo. Thus, the reductions in plasma choline associated with strenuous exercise may reduce acetylcholine release, and could thereby affect endurance or performance.

  15. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

    PubMed Central

    Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M

    2013-01-01

    Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases. PMID:24179466

  16. Homology modeling of human muscarinic acetylcholine receptors.

    PubMed

    Thomas, Trayder; McLean, Kimberley C; McRobb, Fiona M; Manallack, David T; Chalmers, David K; Yuriev, Elizabeth

    2014-01-27

    We have developed homology models of the acetylcholine muscarinic receptors M₁R-M₅R, based on the β₂-adrenergic receptor crystal as the template. This is the first report of homology modeling of all five subtypes of acetylcholine muscarinic receptors with binding sites optimized for ligand binding. The models were evaluated for their ability to discriminate between muscarinic antagonists and decoy compounds using virtual screening using enrichment factors, area under the ROC curve (AUC), and an early enrichment measure, LogAUC. The models produce rational binding modes of docked ligands as well as good enrichment capacity when tested against property-matched decoy libraries, which demonstrates their unbiased predictive ability. To test the relative effects of homology model template selection and the binding site optimization procedure, we generated and evaluated a naïve M₂R model, using the M₃R crystal structure as a template. Our results confirm previous findings that binding site optimization using ligand(s) active at a particular receptor, i.e. including functional knowledge into the model building process, has a more pronounced effect on model quality than target-template sequence similarity. The optimized M₁R-M₅R homology models are made available as part of the Supporting Information to allow researchers to use these structures, compare them to their own results, and thus advance the development of better modeling approaches.

  17. Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.

    PubMed

    Adesioye, Fiyinfoluwa A; Makhalanyane, Thulani P; Biely, Peter; Cowan, Don A

    2016-11-01

    Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect. Such ambiguities can lead to mis-assignment of genes and enzymes during sequence data-mining, reinforcing the necessity for the experimental confirmation of the functional properties of putative AcXE-encoding gene products. Although one-third of all characterized CEs within CAZy families 1-7 and 16 are AcXEs, there is a need to expand the sequence database in order to strengthen the link between AcXE gene sequence and specificity. Currently, most AcXEs are derived from a limited range of (mostly microbial) sources and have been identified via culture-based bioprospecting methods, restricting current knowledge of AcXEs to data from relatively few microbial species. More recently, the successful identification of AcXEs via genome and metagenome mining has emphasised the huge potential of culture-independent bioprospecting strategies. We note, however, that the functional metagenomics approach is still hampered by screening bottlenecks. The most relevant recent reviews of AcXEs have focused primarily on the biochemical and functional properties of these enzymes. In this review, we focus on AcXE phylogeny, classification and the future of metagenomic bioprospecting for novel AcXEs.

  18. Acetylcholine and choline levels in rabbit fetuses exposed to anticholinergics.

    PubMed

    McBride, W G; Hicks, L J

    1987-01-01

    It has been hypothesized that acetylcholine, choline acetylase and acetylcholinesterase may have an ontogenic and trophic influence in the embryo, and that therefore certain drugs may produce malformations via their effect on the acetylcholine and choline levels in the fetus. Thalidomide and the anticholinergics, scopolamine hydrobromide and orphenadrine hydrochloride, and doxylamine succinate, an antihistamine with secondary anticholinergic action, were administered to pregnant New Zealand White rabbit does from day 8 to day 15 of gestation. Cesarean sections were performed on gestational day 16, the fetuses removed and the acetylcholine and choline contents of the fetuses and placentas were estimated by organic extraction and derivation for injection into a GCMS. These acetylcholine and choline levels were compared with those of the fetuses and placentas of the control animals mated with the same buck on the same day as the treated animals. Thalidomide (50 mg/kg) did not affect acetylcholine or choline levels in the fetuses or the placentas obtained from the treated animal. Scopolamine (approximately 100 micrograms/kg) reduced the choline level in the placenta and fetus but not the acetylcholine levels. Orphenadrine (approximately 24 mg/kg) reduced acetylcholine and choline levels in the fetus and choline levels in the placenta. Doxylamine succinate (10 mg/kg) reduced the acetylcholine levels in the fetus and the choline levels in the placenta. The placenta is a fetal organ and the significance of acetylcholine production by the placenta is as yet unknown. The reduction in acetylcholine levels in the fetus exposed to drugs with an anticholinergic action may be of significance in the production of malformations.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Identification of two feruloyl esterases in Dickeya dadantii 3937 and induction of the major feruloyl esterase and of pectate lyases by ferulic acid.

    PubMed

    Hassan, Susan; Hugouvieux-Cotte-Pattat, Nicole

    2011-02-01

    The plant-pathogenic bacterium Dickeya dadantii (formerly Erwinia chrysanthemi) produces a large array of plant cell wall-degrading enzymes. Using an in situ detection test, we showed that it produces two feruloyl esterases, FaeD and FaeT. These enzymes cleave the ester link between ferulate and the pectic or xylan chains. FaeD and FaeT belong to the carbohydrate esterase family CE10, and they are the first two feruloyl esterases to be identified in this family. Cleavage of synthetic substrates revealed strong activation of FaeD and FaeT by ferulic acid. The gene faeT appeared to be weakly expressed, and its product, FaeT, is a cytoplasmic protein. In contrast, the gene faeD is strongly induced in the presence of ferulic acid, and FaeD is an extracellular protein secreted by the Out system, responsible for pectinase secretion. The product of the adjacent gene faeR is involved in the positive control of faeD in response to ferulic acid. Moreover, ferulic acid acts in synergy with polygalacturonate to induce pectate lyases, the main virulence determinant of soft rot disease. Feruloyl esterases dissociate internal cross-links in the polysaccharide network of the plant cell wall, suppress the polysaccharide esterifications, and liberate ferulic acid, which contributes to the induction of pectate lyases. Together, these effects of feruloyl esterases could facilitate soft rot disease caused by pectinolytic bacteria.

  20. Production of cellulolytic enzymes containing cinnamic acid esterase from Schizophyllum commune.

    PubMed

    Tsujiyama, Sho-ichi; Ueno, Hitomi

    2011-01-01

    To develop enzyme preparations capable of digesting plant biomass, we examined the production of cinnamic acid esterase as well as cellulolytic and xylanolytic enzymes in cultures of Schizophyllum commune. The cinnamic acid esterase was produced in the cultures containing solid cellulosic substrates, with production being enhanced by delignifying the wood powder. This indicates that these esterases are produced by cellulose, despite their substrates being phenolic compounds. Cellulolytic and xylanolytic enzymes, with the exception of α-arabinofuranosidase, were also produced in cultures containing cellulosic substances. These results show that enzyme preparation can have high activity of cinnamic acid esterase and cellulolytic and xylanolytic enzymes when S. commune is incubated in the presence of cellulose. These enzyme preparations will be useful for digesting plant biomass and for releasing cinnamic acid derivatives from plant cell walls.

  1. Detection of basal acetylcholine release in the microdialysis of rat frontal cortex by high-performance liquid chromatography using a horseradish peroxidase-osmium redox polymer electrode with pre-enzyme reactor.

    PubMed

    Kato, T; Liu, J K; Yamamoto, K; Osborne, P G; Niwa, O

    1996-06-28

    To determine the basal acetylcholine level in the dialysate of rat frontal cortex, a horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) was employed instead of the conventional platinum electrode used in high-performance liquid chromatography-electrochemical detection (HPLC-ED). In initial experiments, an oxidizable unknown compound interfered with the detection of basal acetylcholine release on HPLC-HRP-GCE. An immobilized peroxidase-choline oxidase precolumn (pre-reactor) was included in the HPLC system, to eliminate the interference from the unknown compound. This combination could detect less than 10 fmol of standard acetylcholine and basal acetylcholine levels in the dialysate from a conventional concentric design microdialysis probe, without the use of cholinesterase inhibitor, and may facilitate physiological investigation of cholinergic neuronal activity in the central nervous system.

  2. A novel protein from mung bean hypocotyl cell walls with acetyl esterase activity.

    PubMed

    Bordenave, M; Goldberg, R; Huet, J C; Pernollet, J C

    1995-01-01

    An acetyl esterase was purified from cell walls isolated from mung bean hypocotyls. The purified enzyme had an apparent Mr of 43,300 and an apparent pI > 9. It rapidly deesterified triacetin and p-nitrophenylacetate and slowly released acetate from beet and flax pectins, the deesterification rate being increased by previous demethylation of the pectins. No significant peptide sequence identity between the acetyl esterase and any known protein could be found in protein data bases.

  3. Flavanone Glycosides as Acetylcholinesterase Inhibitors: Computational and Experimental Evidence

    PubMed Central

    Remya, C.; Dileep, K. V.; Tintu, I.; Variyar, E. J.; Sadasivan, C.

    2014-01-01

    Acetylcholinesterase hydrolyzes the neurotransmitter called acetylcholine and is crucially involved in the regulation of neurotransmission. One of the observable facts in the neurodegenerative disorders like Alzheimer's disease is the decrease in the level of acetylcholine. Available drugs that are used for the treatment of Alzheimer's disease are primarily acetylcholinesterase inhibitors with multiple activities. They maintain the level of acetylcholine in the brain by inhibiting the acetylcholinesterase function. Hence acetylcholinesterase inhibitors can be used as lead compounds for the development of drugs against AD. In the present study, the binding potential of four flavanone glycosides such as naringin, hesperidin, poncirin and sakuranin against acetylcholinesterase was analysed by using the method of molecular modeling and docking. The activity of the top scored compound, naringin was further investigated by enzyme inhibition studies and its inhibitory concentration (IC50) towards acetylcholinesterase was also determined. PMID:25593395

  4. Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection

    PubMed Central

    Rommel, Frank R.; Raghavan, Badrinarayanan; Paddenberg, Renate; Kummer, Wolfgang; Tumala, Susanne; Lochnit, Günter; Gieler, Uwe

    2015-01-01

    Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended. PMID:25673288

  5. Esterase in Imported Fire Ants, Solenopsis invicta and S. richteri (Hymenoptera: Formicidae): Activity, Kinetics and Variation

    PubMed Central

    Chen, J.; Rashid, T.; Feng, G.

    2014-01-01

    Solenopsis invicta and Solenopsis richteri are two closely related invasive ants native to South America. Despite their similarity in biology and behavior, S. invicta is a more successful invasive species. Toxic tolerance has been found to be important to the success of some invasive species. Esterases play a crucial role in toxic tolerance of insects. Hence, we hypothesized that the more invasive S. invicta would have a higher esterase activity than S. richteri. Esterase activities were measured for workers and male and female alates of both ant species using α-naphthyl acetate and β-naphthyl acetate as substrates. Esterase activities in S. invicta were always significantly higher than those in S. richteri supporting our hypothesis. In S. invicta, male alates had the highest esterase activities followed by workers then female alates for both substrates. In S. richetri, for α-naphthyl acetate, male alates had the highest activity followed by female alates then workers, while for β-naphthyl acetate, female alates had the highest activity followed by male alates then workers. For workers, S. richteri showed significantly higher levels of variation about the mean esterase activity than S. invicta. However, S. invicta showed significantly higher levels of variation in both female and male alates. PMID:25408118

  6. Production and purification of a solvent-resistant esterase from Bacillus licheniformis S-86.

    PubMed

    Torres, Sebastián; Baigorí, Mario D; Pandey, Ashok; Castro, Guillermo R

    2008-12-01

    New thermophilic and organic-solvent-tolerant Bacillus licheniformis S-86 strain is able to produce two active and solvent-stable esterases. Production of type I and II esterases was substantially enhanced when oils and surfactants were supplied as carbon sources. Grape oil (0.1% v/v) and Tween 20 to 60 (0.1% v/v) had enhanced enzyme production between 1.6- and 2.2-folds. Type II esterase was purified to homogeneity in a five-step procedure. This esterase was purified 76.7-fold with a specific activity of 135 U mg(-1). Molecular mass of the enzyme was estimated to be 38.4 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Type II esterase was active mostly on esters with short acyl chains, which allowed to classify the enzyme as a carboxylesterase with a K (m) of 80.2 mmol l(-1) and a V (max) of 256.4 micromol min(-1) mg(-1) for p-nitrophenyl acetate. Also, B. licheniformis S-86 type II esterase displayed activity in presence of water-miscible organic solvents at 50% concentration and stability after 1-h incubation.

  7. A stereoselective esterase from Bacillus megaterium: purification, gene cloning, expression and catalytic properties.

    PubMed

    Zheng, Jian-Yong; Wang, Jian; Zhou, Sha-Sha; Li, Xiao-Jun; Ying, Xiang-Xian; Wang, Zhao

    2015-10-27

    Esterases (EC 3.1.1.X) have been used as biocatalysts due to their good stability, high chemo-, regio- and stereoselectivity. In our previous studies, Bacillus megaterium WZ009 harboring esterase displayed the unique capability to convert (S)-4-Chloro-3-hydroxyethylbutyrate (CHBE) in the racemate to (S)-3-hydroxy-γ-butyrolactone (HL) through stereoselective hydrolysis, dechlorination, and lactonization. The remaining (R)-CHBE and formed (S)-HL could be obtained in a one-pot enzymatic reaction. An esterase from B. megaterium WZ009 was purified and was found to have 466 encoded amino acids and an apparent molecular mass of 55 kDa. The purified esterase exhibited maximal activity at a temperature of 25 °C and at a pH of 11.5 towards 100 mM CHBE. When the stereoselective biocatalysis of rac-CHBE was performed using the recombinant Escherichia coli BL21 (DH3) cells harboring the esterase, the catalytic activity increased by 20-fold compared with the original strain B. megaterium WZ009. With the addition of activated carbon (62 g/L) in the reaction system, the conversion was increased from 39% to 45% at a substrate concentration of 750 mM. Another remarkable advantage is that both of the obtained residual (R)-CHBE and the formed (S)-HL had high optical purities (e.e.s > 99.9%, e.e.p > 99.9%), thereby making this esterase a usable biocatalyst for industrial application.

  8. Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila

    PubMed Central

    Younus, Faisal; Fraser, Nicholas J.; Coppin, Chris W.; Liu, Jian-Wei; Correy, Galen J.; Chertemps, Thomas; Pandey, Gunjan; Maïbèche, Martine; Jackson, Colin J.; Oakeshott, John G.

    2017-01-01

    Previous electrophysiological and behavioural studies implicate esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. Here we show esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. Our data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate. PMID:28393888

  9. Acetylcholine receptors in the human retina

    SciTech Connect

    Hutchins, J.B.; Hollyfield, J.G.

    1985-11-01

    Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand TH-propylbenzilylcholine mustard (TH-PrBCM) to label muscarinic receptors. TH- or SVI-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that TH-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer of the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina.

  10. Impulsive behavior and nicotinic acetylcholine receptors.

    PubMed

    Ohmura, Yu; Tsutsui-Kimura, Iku; Yoshioka, Mitsuhiro

    2012-01-01

    Higher impulsivity is thought to be a risk factor for drug addiction, criminal involvement, and suicide. Excessive levels of impulsivity are often observed in several psychiatric disorders including attention-deficit/hyperactivity disorder and schizophrenia. Previous studies have demonstrated that nicotinic acetylcholine receptors (nAChRs) are involved in impulsive behavior. Here, we introduce recent advances in this field and describe the role of the following nAChR-related brain mechanisms in modulating impulsive behavior: dopamine release in the ventral striatum; α4β2 nAChRs in the infralimbic cortex, which is a ventral part of the medial prefrontal cortex (mPFC); and dopamine release in the mPFC. We also suggest several potential therapeutic drugs to address these mechanisms in impulsivity-related disorders and explore future directions to further elucidate the roles of central nAChRs in impulsive behavior.

  11. Alcohol's actions on neuronal nicotinic acetylcholine receptors.

    PubMed

    Davis, Tiffany J; de Fiebre, Christopher M

    2006-01-01

    Although it has been known for many years that alcoholism and tobacco addiction often co-occur, relatively little information is available on the biological factors that regulate the co-use and abuse of nicotine and alcohol. In the brain, nicotine acts at several different types of receptors collectively known as nicotinic acetylcholine receptors (nAChRs). Alcohol also acts on at least some of these receptors, enhancing the function of some nAChR subtypes and inhibiting the activity of others. Chronic alcohol and nicotine administration also lead to changes in the numbers of nAChRs. Natural variations (i.e., polymorphisms) in the genes encoding different nAChR subunits may be associated with individual differences in the sensitivity to some of alcohol's and nicotine's effects. Finally, at least one subtype of nAChR may help protect cells against alcohol-induced neurotoxicity.

  12. Conotoxins Targeting Nicotinic Acetylcholine Receptors: An Overview

    PubMed Central

    Lebbe, Eline K. M.; Peigneur, Steve; Wijesekara, Isuru; Tytgat, Jan

    2014-01-01

    Marine snails of the genus Conus are a large family of predatory gastropods with an unparalleled molecular diversity of pharmacologically active compounds in their venom. Cone snail venom comprises of a rich and diverse cocktail of peptide toxins which act on a wide variety of ion channels such as voltage-gated sodium- (NaV), potassium- (KV), and calcium- (CaV) channels as well as nicotinic acetylcholine receptors (nAChRs) which are classified as ligand-gated ion channels. The mode of action of several conotoxins has been the subject of investigation, while for many others this remains unknown. This review aims to give an overview of the knowledge we have today on the molecular pharmacology of conotoxins specifically interacting with nAChRs along with the structure–function relationship data. PMID:24857959

  13. [Desensitization of the nicotinic acetylcholine receptor].

    PubMed

    Quiñonez, M; Rojas, L

    1994-01-01

    In biological membranes, ionic channels act speeding up ion movements. Each ionic channel is excited by a specific stimulus (i.e. electric, mechanical, chemical, etc.). Chemically activated ionic channels (CAIC), such as the nicotinic acetylcholine receptor (nAChR), suffer desensitization when the receptor site is still occupied by the agonist molecule. The desensitized CAIC is a non functional channel state regarded as a particular case of receptors rundown. CAIC desensitization only involve reduced activity and not their membrane elimination. Desensitization is important to control synaptic transmission and the development of the nervous system. In this review we discuss results related to its production, modulation and some aspects associated to models that consider it. Finally, an approach combining molecular biology and electrophysiology techniques to understand desensitization and its importance in biological systems is presented.

  14. Some Anti-Inflammatory Agents Inhibit Esterase Activities of Human Carbonic Anhydrase Isoforms I and II: An In Vitro Study.

    PubMed

    Alım, Zuhal; Kılınç, Namık; İşgör, Mehmet M; Şengül, Bülent; Beydemir, Şükrü

    2015-10-01

    Carbonic anhydrases (CAs) are known as a drug-target enzymes. The inhibitors of the enzyme are important compounds for discovering new therapeutic agents and understanding in detail protein-drug interactions at the molecular level. For this purpose, the in vitro effects of some anti-inflammatory agents such as tenoxicam, fluorometholone acetate, and dexamethasone were investigated on esterase activity of human erythrocyte CA-I and CA-II in this study. hCA-I and hCA-II were purified by affinity chromatography with a yield of 47.25% and 87%, and a specific activity of 642.8 EU/mg proteins and 5576.9 EU/mg proteins, respectively. SDS-PAGE was performed to determine the purity of the enzymes. Inhibitory effects of the drugs on hCA-I and hCA-II were determined by spectrophotometric method. IC50 values for hCA-I and hCA-II were 0.198, 2.18, 11.7, 0.11, 17.5 and 14 μm using tenoxicam, fluorometholone acetate, and dexamethasone, respectively. For fluorometholone acetate and dexamethasone, Ki values from Lineweaver-Burk plots were obtained as 1.044 and 21.2 μm (noncompetitive) for hCA-I and 9.98 and 8.66 μm (non-competitive) for hCA-II. In conclusion, tenoxicam, fluorometholone acetate, and dexamethasone showed potent inhibitory effects on esterase activity of hCA-I and hCA-II isozymes under in vitro conditions.

  15. "Warming yang and invigorating qi" acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis.

    PubMed

    Huang, Hai-Peng; Pan, Hong; Wang, Hong-Feng

    2016-03-01

    Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. "Warming yang and invigorating qi" acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following "warming yang and invigorating qi" acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10), Zusanli (ST36), Pishu (BL20), and Shenshu (BL23) once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that "warming yang and invigorating qi" acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis.

  16. Modulation of nicotinic acetylcholine receptors by strychnine

    PubMed Central

    García-Colunga, Jesús; Miledi, Ricardo

    1999-01-01

    Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex. PMID:10097172

  17. [New anticoagulants - direct thrombin inhibitors].

    PubMed

    Brand, B; Graf, L

    2012-11-01

    Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other.

  18. Is the acetylcholine receptor a rabies virus receptor?

    PubMed

    Lentz, T L; Burrage, T G; Smith, A L; Crick, J; Tignor, G H

    1982-01-08

    Rabies virus was found on mouse diaphragms and on cultured chick myotubes in a distribution coinciding with that of the acetylcholine receptor. Treatment of the myotubes with alpha-bungarotoxin and d-tubocurarine before the addition of the virus reduced the number of myotubes that became infected with rabies virus. These findings together suggest that acetylcholine receptors may serve as receptors for rabies virus. The binding of virus to acetylcholine receptors, which are present in high density at the neuromuscular junction, would provide a mechanism whereby the virus could be locally concentrated at sites in proximity to peripheral nerves facilitating subsequent uptake and transfer to the central nervous system.

  19. Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase

    SciTech Connect

    Yin, D.; Bernhardt, P; Morley, K; Jiang, Y; Cheeseman, J; Purpero, V; Schrag, J; Kazlauskas, R

    2010-01-01

    Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of {var_epsilon}-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k{sub cat}, but K{sub m} also increased so the specificity constant, k{sub cat}/K{sub m}, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of {var_epsilon}-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the

  20. Acetylcholine released from cholinergic nerves contributes to cutaneous vasodilation during heat stress

    NASA Technical Reports Server (NTRS)

    Shibasaki, Manabu; Wilson, Thad E.; Cui, Jian; Crandall, Craig G.

    2002-01-01

    Nitric oxide (NO) contributes to active cutaneous vasodilation during a heat stress in humans. Given that acetylcholine is released from cholinergic nerves during whole body heating, coupled with evidence that acetylcholine causes vasodilation via NO mechanisms, it is possible that release of acetylcholine in the dermal space contributes to cutaneous vasodilation during a heat stress. To test this hypothesis, in seven subjects skin blood flow (SkBF) and sweat rate were simultaneously monitored over three microdialysis membranes placed in the dermal space of dorsal forearm skin. One membrane was perfused with the acetylcholinesterase inhibitor neostigmine (10 microM), the second membrane was perfused with the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME; 10 mM) dissolved in the aforementioned neostigmine solution (l-NAME(Neo)), and the third membrane was perfused with Ringer solution as a control site. Each subject was exposed to approximately 20 min of whole body heating via a water-perfused suit, which increased mean body temperature from 36.4 +/- 0.1 to 37.5 +/- 0.1 degrees C (P < 0.05). After the heat stress, SkBF at each site was normalized to its maximum value, identified by administration of 28 mM sodium nitroprusside. Mean body temperature threshold for cutaneous vasodilation was significantly lower at the neostigmine-treated site relative to the other sites (neostigmine: 36.6 +/- 0.1 degrees C, l-NAME(Neo): 37.1 +/- 0.1 degrees C, control: 36.9 +/- 0.1 degrees C), whereas no significant threshold difference was observed between the l-NAME(Neo)-treated and control sites. At the end of the heat stress, SkBF was not different between the neostigmine-treated and control sites, whereas SkBF at the l-NAME(Neo)-treated site was significantly lower than the other sites. These results suggest that acetylcholine released from cholinergic nerves is capable of modulating cutaneous vasodilation via NO synthase mechanisms early in the heat stress but

  1. Organophosphates induce distal axonal damage, but not brain oedema, by inactivating neuropathy target esterase

    SciTech Connect

    Read, David J.; Li Yong; Chao, Moses V.; Cavanagh, John B.; Glynn, Paul

    2010-05-15

    Single doses of organophosphorus compounds (OP) which covalently inhibit neuropathy target esterase (NTE) can induce lower-limb paralysis and distal damage in long nerve axons. Clinical signs of neuropathy are evident 3 weeks post-OP dose in humans, cats and chickens. By contrast, clinical neuropathy in mice following acute dosing with OPs or any other toxic compound has never been reported. Moreover, dosing mice with ethyloctylphosphonofluoridate (EOPF) - an extremely potent NTE inhibitor - causes a different (subacute) neurotoxicity with brain oedema. These observations have raised the possibility that mice are intrinsically resistant to neuropathies induced by acute toxic insult, but may incur brain oedema, rather than distal axonal damage, when NTE is inactivated. Here we provide the first report that hind-limb dysfunction and extensive axonal damage can occur in mice 3 weeks after acute dosing with a toxic compound, bromophenylacetylurea. Three weeks after acutely dosing mice with neuropathic OPs no clinical signs were observed, but distal lesions were present in the longest spinal sensory axons. Similar lesions were evident in undosed nestin-cre:NTEfl/fl mice in which NTE had been genetically-deleted from neural tissue. The extent of OP-induced axonal damage in mice was related to the duration of NTE inactivation and, as reported in chickens, was promoted by post-dosing with phenylmethanesulfonylfluoride. However, phenyldipentylphosphinate, another promoting compound in chickens, itself induced in mice lesions different from the neuropathic OP type. Finally, EOPF induced subacute neurotoxicity with brain oedema in both wild-type and nestin-cre:NTEfl/fl mice indicating that the molecular target for this effect is not neural NTE.

  2. Leukocyte esterase-nitrite and bioluminescence assays as urine screens.

    PubMed Central

    Males, B M; Bartholomew, W R; Amsterdam, D

    1985-01-01

    The 1-min leukocyte esterase (LE)-nitrite test (Chemstrip 9; Biodynamics, Division of Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and a bioluminescence assay (Monolight centrifugation method; Analytical Luminescence Laboratory, Inc., San Diego, Calif.) were tested for their efficacy as urine screens among 453 patients at a tertiary-care teaching hospital. Both methods had the capacity to exclude significant bacteriuria (greater than or equal to 10(5) CFU/ml) when compared with the results of conventional culture methods, with predictive values of 99 and 93%, respectively, for a negative test. Bioluminescence was the more accurate nonculture method used. Sensitivity and specificity values were 97 and 71%, respectively, for bioluminescence, 82 and 60%, respectively, for LE with nitrite, and 72 and 64%, respectively, for LE without nitrite. At reduced levels of bacteriuria less than 10(5) CFU/ml), the sensitivities of LE-nitrite and bioluminescence were decreased but comparable. The addition of protein and blood test results in the Chemstrip 9, along with LE-nitrite as bacteriuria indicators, were unsatisfactory because of the large numbers of false-positive results attributed to protein and blood determinations. LE activity as detected by the LE test was a poor predictor of significant bacteriuria in both male and female patients. The sensitivity (71%) and specificity (57%) of the LE test in male patients were significantly lower than those previously reported and varied with the patient population studied. PMID:3935662

  3. Hydrolysis of synthetic polyesters by Clostridium botulinum esterases.

    PubMed

    Perz, Veronika; Baumschlager, Armin; Bleymaier, Klaus; Zitzenbacher, Sabine; Hromic, Altijana; Steinkellner, Georg; Pairitsch, Andris; Łyskowski, Andrzej; Gruber, Karl; Sinkel, Carsten; Küper, Ulf; Ribitsch, Doris; Guebitz, Georg M

    2016-05-01

    Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.

  4. Lipases and esterases from extremophiles: overview and case example of the production and purification of an esterase from Thermus thermophilus HB27.

    PubMed

    Fuciños, Pablo; González, Roberto; Atanes, Estrella; Sestelo, Ana Belén Fernández; Pérez-Guerra, Nelson; Pastrana, Lorenzo; Rúa, María Luisa

    2012-01-01

    Extremophiles are organisms that have evolved to exist in a variety of extreme environments. They fall into a number of different classes that include thermophiles, halophiles, acidophiles, alkalophiles, psychrophiles, and barophiles (piezophiles). Extremophiles have the potential to produce uniquely valuable biocatalysts that function under conditions in which usually the enzymes of their nonextremophilic counterparts could not. Among novel enzymes isolated from extremophilic microorganisms, hydrolases, and particularly lipases and esterases are experiencing a growing demand. Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) catalyze the cleavage of ester bounds in aqueous media and the reverse reaction in organic solvents. Both lipolytic enzymes have relevant applications in food, dairy, detergent, biofuel, and pharmaceutical industries. Here, we summarize the properties of lipases and esterases from the main extremophile groups: thermophiles and hyperthermophiles, psychrophiles, halophiles, alkalophiles/acidophiles, and solvent-resistant microorganisms.We report the biomass and lipolytic activity production by Thermus thermophilus HB27 in 5-L stirred-tank bioreactor at 70°C. Suitability of thermal spring water for culture media formulation is shown. In addition, a protocol to isolate and purify a cell-bound esterase from this microorganism is described.

  5. Deacetylation of sialic acid by esterases potentiates pneumococcal neuraminidase activity for mucin utilization, colonization and virulence

    PubMed Central

    Kahya, Hasan F.; Andrew, Peter W.

    2017-01-01

    Pneumococcal neuraminidase is a key enzyme for sequential deglycosylation of host glycans, and plays an important role in host survival, colonization, and pathogenesis of infections caused by Streptococcus pneumoniae. One of the factors that can affect the activity of neuraminidase is the amount and position of acetylation present in its substrate sialic acid. We hypothesised that pneumococcal esterases potentiate neuraminidase activity by removing acetylation from sialic acid, and that will have a major effect on pneumococcal survival on mucin, colonization, and virulence. These hypotheses were tested using isogenic mutants and recombinant esterases in microbiological, biochemical and in vivo assays. We found that pneumococcal esterase activity is encoded by at least four genes, SPD_0534 (EstA) was found to be responsible for the main esterase activity, and the pneumococcal esterases are specific for short acyl chains. Assay of esterase activity by using natural substrates showed that both the Axe and EstA esterases could use acetylated xylan and Bovine Sub-maxillary Mucin (BSM), a highly acetylated substrate, but only EstA was active against tributyrin (triglyceride). Incubation of BSM with either Axe or EstA led to the acetate release in a time and concentration dependent manner, and pre-treatment of BSM with either enzyme increased sialic acid release on subsequent exposure to neuraminidase A. qRT-PCR results showed that the expression level of estA and axe increased when exposed to BSM and in respiratory tissues. Mutation of estA alone or in combination with nanA (codes for neuraminidase A), or the replacement of its putative serine active site to alanine, reduced the pneumococcal ability to utilise BSM as a sole carbon source, sialic acid release, colonization, and virulence in a mouse model of pneumococcal pneumonia. PMID:28257499

  6. Pharmacological profile of zacopride and new quaternarized fluorobenzamide analogues on mammalian α7 nicotinic acetylcholine receptor.

    PubMed

    Bourdin, Céline M; Lebreton, Jacques; Mathé-Allainmat, Monique; Thany, Steeve H

    2015-08-15

    From quaternarization of quinuclidine enantiomers of 2-fluoro benzamide LMA10203 in dichloromethane, the corresponding N-chloromethyl derivatives LMA10227 and LMA10228 were obtained. Here, we compared the agonist action of known zacopride and its 2-fluoro benzamide analogues, LMA10203, LMA10227 and LMA10228 against mammalian homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We found that LMA10203 was a partial agonist of α7 receptor with a pEC50 value of 4.25 ± 0.06 μM whereas LMA10227 and LMA10228 were poorly active on α7 homomeric nicotinic receptor. LMA10227 and LMA10228 were identified as antagonists of acetylcholine-induced currents with IC50 values of 28.4 μM and 39.3 μM whereas LMA10203 and zacopride possessed IC50 values of 8.07 μM and 7.04 μM, respectively. Moreover, despite their IC50 values, LMA10227 was the most potent inhibitor of nicotine-induced current amplitudes (65.7 ± 2.1% inhibition). LMA10203 and LMA10228 had the same inhibitory effects (26.5 ± 7.5% and 33.2 ± 4.1%, respectively), whereas zacopride had no significant inhibitory effect (4.37 ± 4%) on nicotine-induced responses. Our results revealed different pharmacological properties between the four compounds on acetylcholine and nicotine currents. The mode of action of benzamide compounds may need to be reinterpreted with respect to the potential role of α7 receptor.

  7. Modal gating of muscle nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Vij, Ridhima

    Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that

  8. Acetylcholine Promotes Binding of α-Conotoxin MII for α3β2 Nicotinic Acetylcholine Receptors

    PubMed Central

    Sambasivarao, Somisetti V.; Roberts, Jessica; Bharadwaj, Vivek S.; Slingsby, Jason G.; Rohleder, Conrad; Mallory, Chris; Groome, James R.

    2014-01-01

    α-Conotoxin MII (α-CTxMII) is a 16 amino acid peptide with the sequence GCCSNPVCHLEHSNLC containing disulfide bonds between Cys2-Cys8 and Cys3-Cys16. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand binding affinity, homology models of the heteropentameric α3β2-nAChR were constructed. The models were created in MODELLER using crystal structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3 and β2 subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with commensurate binding energies to previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3β2-nAChR for α-CTxMII with ACh bound to the receptor, which was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs. PMID:24420650

  9. Captopril augments acetylcholine-induced bronchial smooth muscle contractions in vitro via kinin-dependent mechanisms.

    PubMed

    Agrawal, Naman; Akella, Aparna; Deshpande, Shripad B

    2016-06-01

    Angiotensin converting enzyme (ACE) inhibitors therapy is aassociated with bothersome dry cough as an adverse effect. The mechanisms underlying this adverse effect are not clear. Therefore, influence of captopril (an ACE inhibitor) on acetylcholine (ACh)-induced bronchial smooth muscle contractions was investigated. Further, the mechanisms underlying the captopril-induced changes were also explored. In vitro contractions of rat bronchial smooth muscle to cumulative concentrations of ACh were recorded before and after exposure to captopril. Further, the involvement of kinin and inositol triphosphate (IP₃) pathways for captopril-induced alterations were explored. ACh produced concentration-dependent (5-500 µM) increase in bronchial smooth muscle contractions. Pre-treatment with captopril augmented the ACh-induced contractions at each concentration significantly. Pre-treatment with aprotinin (kinin synthesis inhibitor) or heparin (inositol triphosphate, IP₃-inhibitor), blocked the captopril-induced augmentation of bronchial smooth muscle contractions evoked by ACh. Further, captopril-induced augmentation was absent in calcium-free medium. These results suggest that captopril sensitizes bronchial smooth muscles to ACh-induced contractions. This sensitization may be responsible for dry cough associated with captopril therapy.

  10. Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

    PubMed

    Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

    2015-07-01

    Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

  11. Esterase Active in Polar Organic Solvents from the Yeast Pseudozyma sp. NII 08165

    PubMed Central

    Shainu, Anju; Pandey, Ashok; Sukumaran, Rajeev K.

    2014-01-01

    Esterases/lipases active in water miscible solvents are highly desired in biocatalysis where substrate solubility is limited and also when the solvent is desired as an acyl acceptor in transesterification reactions, as with the case of biodiesel production. We have isolated an esterase from the glycolipid producing yeast-Pseudozyma sp. NII 08165 which in its crude form was alkali active, thermo stable, halo tolerant and also capable of acting in presence of high methanol concentration. The crude enzyme which maintained 90% of its original activity after being treated at 70°C was purified and the properties were characterized. The partially purified esterase preparation had temperature and pH optima of 60°C and 8.0 respectively. The enzyme retained almost complete activity in presence of 25% methanol and 80% activity in the same strength of ethanol. Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield. One of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized. Purified LIP1 had a Km and Vmax of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol. PMID:24800063

  12. Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

    PubMed

    Martin del Campo, Martha; Camacho, Rosa M; Mateos-Díaz, Juan C; Müller-Santos, Marcelo; Córdova, Jesus; Rodríguez, Jorge A

    2015-11-01

    Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

  13. Organophosphate and Pyrethroid Hydrolase Activities of Mutant Esterases from the Cotton Bollworm Helicoverpa armigera

    PubMed Central

    Li, Yongqiang; Farnsworth, Claire A.; Coppin, Chris W.; Teese, Mark G.; Liu, Jian-Wei; Scott, Colin; Zhang, Xing; Russell, Robyn J.; Oakeshott, John G.

    2013-01-01

    Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes’ active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4–6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively. PMID:24204917

  14. Molecular and kinetic evidence for allelic variants of esterase Estbeta1 in the mosquito Culex quinquefasciatus.

    PubMed

    Small, G J; Karunaratne, S H; Chadee, D D; Hemingway, J

    1999-07-01

    Elevated esterase Estbeta1 was purified from larvae of newly isolated strains of the mosquito Culex quinquefasciatus from Colombia (COL) and Trinidad (TRI) with resistance to organophosphate (OP) insecticides. Insecticide interactions were compared with those of elevated Estbeta1(2) from the OP-resistant Habana strain and the non-elevated Estbeta1(3) from the susceptible PelSS strain. On the basis of insecticide binding efficiency, all elevated Estbeta1 esterases were readily distinguishable. Differences between the EcoRI restriction fragment patterns of the amplified estbeta1 gene in COL and TRI strains compared with each other, and between amplified estbeta1(1), estbeta1(2) and the non-amplified estbeta1(3), suggest differences in their nucleotide sequence. Considering their variable insecticide binding efficiencies, these genetic differences would imply that, in contrast to estalpha2 and estbeta2, amplification of estbeta1 has occurred several times independently. Generally, the elevated Estbeta1s were more reactive with insecticides than the non-elevated Estbeta1(3). This supports the hypothesis that the elevated esterase-based mechanism confers resistance through amplification of alleles coding for esterases which have a greater specificity for the insecticides they sequester than the esterases coded by their non-amplified counterparts.

  15. GLYCOENGINEERING OF ESTERASE ACTIVITY THROUGH METABOLIC FLUX-BASED MODULATION OF SIALIC ACID.

    PubMed

    Mathew, Mohit; Tan, Elaine; Labonte, Jason W; Shah, Shivam; Saeui, Christopher T; Liu, Lingshu; Bhattacharya, Rahul; Bovonratwet, Patawut; Gray, Jeffrey J; Yarema, Kevin

    2017-02-20

    This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of the carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity. By contrast, hexosamine analogs not targeted to sialic acid biosynthesis (e.g., butanoylated GlcNAc or GalNAc) had minimal impact. Measurement of mRNA and protein confirmed that esterase activity was controlled through glycosylation and not through transcription or translation. Azide-modified ManNAc analogs widely used in MGE also enhanced esterase activity and provided a way to enrich targeted "glycoengineered" proteins (such as CES2), thereby providing unambiguous evidence that the compounds were converted to sialosides and installed into the glycan structures of esterases as intended. Overall, this study provides a pioneering example of the modulation of intracellular enzyme activity through MGE, which expands the value of this technology from its current status as a labeling strategy and modulator of cell surface biological events.

  16. Low acetylcholine during slow-wave sleep is critical for declarative memory consolidation.

    PubMed

    Gais, Steffen; Born, Jan

    2004-02-17

    The neurotransmitter acetylcholine is considered essential for proper functioning of the hippocampus-dependent declarative memory system, and it represents a major neuropharmacological target for the treatment of memory deficits, such as those in Alzheimer's disease. During slow-wave sleep (SWS), however, declarative memory consolidation is particularly strong, while acetylcholine levels in the hippocampus drop to a minimum. Observations in rats led to the hypothesis that the low cholinergic tone during SWS is necessary for the replay of new memories in the hippocampus and their long-term storage in neocortical networks. However, this low tone should not affect nondeclarative memory systems. In this study, increasing central nervous cholinergic activation during SWS-rich sleep by posttrial infusion of 0.75 mg of the cholinesterase inhibitor physostigmine completely blocked SWS-related consolidation of declarative memories for word pairs in human subjects. The treatment did not interfere with consolidation of a nondeclarative mirror tracing task. Also, physostigmine did not alter memory consolidation during waking, when the endogenous central nervous cholinergic tone is maximal. These findings are in line with predictions that a low cholinergic tone during SWS is essential for declarative memory consolidation.

  17. Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

    SciTech Connect

    Resende, R.R.; Alves, A.S.; Britto, L.R.G; Ulrich, H.

    2008-04-15

    Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G{alpha}{sub q/11}-coupled M{sub 1}, M{sub 3} and M{sub 5} receptors and intracellular calcium stores, whereas G{alpha}{sub i/o}-protein coupled M{sub 2} receptor activity mediated neuronal differentiation.

  18. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    SciTech Connect

    Miles, K.; Anthony, D.T.; Rubin, L.L.; Greengard, P.; Huganir, R.L.

    1987-09-01

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.

  19. Kinetics of the inhibitory interaction of organophosphorus neuropathy inducers and non-inducers in soluble esterases in the avian nervous system

    SciTech Connect

    Mangas, Iris; Vilanova, Eugenio; Estevez, Jorge

    2011-11-15

    Some published studies suggest that low level exposure to organophosphorus esters (OPs) may cause neurological and neurobehavioral effects at long term exposure. These effects cannot be explained by action on known targets. In this work, the interactions (inhibition, spontaneous reactivation and 'ongoing inhibition') of two model OPs (paraoxon, non neuropathy-inducer, and mipafox, neuropathy-inducer) with the chicken brain soluble esterases were evaluated. The best-fitting kinetic model with both inhibitors was compatible with three enzymatic components. The amplitudes (proportions) of the components detected with mipafox were similar to those obtained with paraoxon. These observations confirm the consistency of the results and the model applied and may be considered an external validation. The most sensitive component (E{alpha}) for paraoxon (11-23% of activity, I{sub 50} (30 min) = 9-11 nM) is also the most sensitive for mipafox (I{sub 50} (30 min) = 4 nM). This component is spontaneously reactivated after inhibition with paraoxon. The second sensitive component to paraoxon (E{beta}, 71-84% of activity; I{sub 50} (30 min) = 1216 nM) is practically resistant to mipafox. The third component (E{gamma}, 5-8% of activity) is paraoxon resistant and has I{sub 50} (30 min) of 3.4 {mu}M with mipafox, similar to NTE (neuropathy target esterase). The role of these esterases remains unknown. Their high sensitivity suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation. They will have to be considered in further metabolic and toxicological studies. -- Research Highlights: Black-Right-Pointing-Pointer Paraoxon and mipafox interactions have been evaluated with chicken soluble brain esterases. Black-Right-Pointing-Pointer The paraoxon inhibition was analyzed considering the simultaneous spontaneous reactivation. Black-Right-Pointing-Pointer The best

  20. Catestatin attenuates endoplasmic reticulum induced cell apoptosis by activation type 2 muscarinic acetylcholine receptor in cardiac ischemia/reperfusion

    PubMed Central

    Liao, Feng; Zheng, Yang; Cai, Junyan; Fan, Jinghui; Wang, Jing; Yang, Jichun; Cui, Qinghua; Xu, Guoheng; Tang, Chaoshu; Geng, Bin

    2015-01-01

    Catestatin (CST) is a catecholamine secretion inhibiting peptide as non-competitive inhibitor of nicotinic acetylcholine receptor. CST play a protective role in cardiac ischemia/reperfusion (I/R) but the molecular mechanism remains unclear. Cardiomyocytes endogenously produced CST and its expression was reduced after I/R. CST pretreatment decreased apoptosis especially endoplasmic reticulum (ER) stress response during I/R. The protection of CST was confirmed in H9c2 cardiomyoblasts under Anoxia/reoxygenation (A/R). In contrast, siRNA-mediated knockdown of CST exaggerated ER stress induced apoptosis. The protective effects of CST were blocked by extracellular signal-regulated kinases 1/2 (ERK1/2) inhibitor PD90895 and phosphoinositide 3-kinase (PI3 K) inhibitor wortmannin. CST also increased ERK1/2 and protein kinase B (Akt) phosphorylation and which was blocked by atropine and selective type 2 muscarinic acetylcholine (M2) receptor, but not type 1 muscarinic acetylcholine (M1) receptor antagonist. Receptor binding assay revealed that CST competitively bound to the M2 receptor with a 50% inhibitory concentration of 25.7 nM. Accordingly, CST inhibited cellular cAMP stimulated by isoproterenol or forskolin, and which was blocked by selective M2 receptor antagonist. Our findings revealed that CST binds to M2 receptor, then activates ERK1/2 and PI3 K/Akt pathway to inhibit ER stress-induced cell apoptosis resulting in attenuation cardiac I/R injury. PMID:26567709

  1. Mycobacteriocins produced by rapidly growing mycobacteria are Tween-hydrolyzing esterases.

    PubMed Central

    Saito, H; Tomioka, H; Watanabe, T; Yoneyama, T

    1983-01-01

    Smegmatocin, a protein produced by Mycobacterium smegmatis ATCC 14468, was found to have an esterase activity, hydrolyzing Tween 80, polyoxyethylene sorbitan monooleate, added to the assay medium for various "bacteriocins" from mycobacteria. Because M. diernhoferi ATCC 19340 (indicator strain for smegmatocin) is highly susceptible to oleic acid and smegmatocin requires Tween 80 for manifestation of its anti-M. diernhoferi activity, it is likely that smegmatocin-mediated antimicrobial action is caused by oleic acid generated by hydrolysis of Tween 80 by the inherent esterase action of smegmatocin. Other mycobacteriocins from rapidly growing mycobacteria also have inherent esterase activity against Tween 80 and require Tween 80 for expression of antimycobacterial action. Smegmatocin was found to hydrolyze various polyoxyethylene (sorbitan) fatty acyl esters but not sorbitan monooleate and glyceryl esters. Images PMID:6826523

  2. Eco-friendly surface modification on polyester fabrics by esterase treatment

    NASA Astrophysics Data System (ADS)

    Wu, Jindan; Cai, Guoqiang; Liu, Jinqiang; Ge, Huayun; Wang, Jiping

    2014-03-01

    Currently, traditional alkali deweighting technology is widely used to improve the hydrophilicity of polyester fabrics. However, the wastewater and heavy chemicals in the effluent cause enormous damage to the environment. Esterase treatment, which is feasible in mild conditions with high selectivity, can provide a clean and efficient way for polyester modification. Under the optimum conditions, the polyester fabric hydrolysis process of esterase had a linear kinetics. X-ray photoelectron spectrometry (XPS) results showed that hydroxyl and carboxyl groups were produced only on the surface of modified fiber without changing the chemical composition of the bulk. These fibers exhibited much improved fabric wicking, as well as greatly improved oily stain removal performance. Compared to the harsh alkali hydrolysis, the enzyme treatment led to smaller weight loss and better fiber integrity. The esterase treatment technology is promising to produce higher-quality polyester textiles with an environmental friendly approach.

  3. Role of an esterase in flavor volatile variation within the tomato clade.

    PubMed

    Goulet, Charles; Mageroy, Melissa H; Lam, Nghi B; Floystad, Abbye; Tieman, Denise M; Klee, Harry J

    2012-11-13

    Tomato flavor is dependent upon a complex mixture of volatiles including multiple acetate esters. Red-fruited species of the tomato clade accumulate a relatively low content of acetate esters in comparison with the green-fruited species. We show that the difference in volatile ester content between the red- and green-fruited species is associated with insertion of a retrotransposon adjacent to the most enzymatically active member of a family of esterases. This insertion causes higher expression of the esterase, resulting in the reduced levels of multiple esters that are negatively correlated with human preferences for tomato. The insertion was evolutionarily fixed in the red-fruited species, suggesting that high expression of the esterase and consequent low ester content may provide an adaptive advantage in the ancestor of the red-fruited species. These results illustrate at a molecular level how closely related species exhibit major differences in volatile production by altering a volatile-associated catabolic activity.

  4. Extraction and purification of wheat-esterase using aqueous two-phase systems of ionic liquid and salt.

    PubMed

    Jiang, Bin; Feng, Zhibiao; Liu, Chunhong; Xu, Yingcao; Li, Dongmei; Ji, Guo

    2015-05-01

    To explore a new and simple rapid extraction and purification technique for wheat-esterase, an ionic liquids (ILs)-based aqueous two-phase system (ATPS) was developed for the purification of wheat-esterase from wheat extracts. Effects of various process parameters such as the concentrations of [Bmim]BF4, the types and concentrations of phase-forming salt, the system pH and the temperature on partitioning of wheat-esterase were evaluated. The obtained data indicated that wheat-esterase was preferentially partitioned into the ILs-rich phase and the ATPS composed of 20 % [Bmim]BF4 (w/w) and 25 % (w/w) NaH2PO4(pH = 4.8) showed good selectivity on wheat-esterase. Under the optimum conditions, wheat-esterase was purified with an acceptable yield (88.93 %), but produced wheat-esterase was 4.23 times as pure. It was obvious that temperature shows little influence on the purification between 10 and 50 °C. Sephadex G-150FF revealed that the band intensity of contaminating proteins in ATPS fraction almost disappeared. Therefore, ILs-based ATPS was an effective method for partitioning and recovery of wheat-esterase from wheat crude extracts.

  5. Acetylcholine receptor binding antibody-associated myasthenia gravis and rhabdomyolysis induced by nivolumab in a patient with melanoma.

    PubMed

    Shirai, Takushi; Sano, Tasuku; Kamijo, Fuminao; Saito, Nana; Miyake, Tomomi; Kodaira, Minori; Katoh, Nagaaki; Nishie, Kenichi; Okuyama, Ryuhei; Uhara, Hisashi

    2016-01-01

    We reported an 81-year-old woman with metastatic melanoma, in whom myasthenia gravis and rhabdomyolysis developed after nivolumab monotherapy. The first symptom of myasthenia gravis was dyspnea. Ultrasonography detected hypokinesis of the bilateral diaphragm suggesting myasthenia gravis, although there was no abnormal finding of the lungs in computed tomography images. Acetylcholine receptor binding antibodies were low-titer positive in the preserved serum before administration of nivolumab, strongly suggesting that the myasthenia gravis was a nivolumab-related immune adverse event. Despite the remarkable clinical benefits of immune checkpoint inhibitors for patients with advanced melanoma, it is important to recognize unexpected immune-related adverse events.

  6. Characterization of a novel cold active and salt tolerant esterase from Zunongwangia profunda.

    PubMed

    Rahman, Mohammad Asadur; Culsum, Umma; Tang, Wenhao; Zhang, Shao Wei; Wu, Gaobing; Liu, Ziduo

    2016-04-01

    A novel cold active esterase, EstLiu was cloned from the marine bacterium Zunongwangia profunda, overexpressed in E. coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography. The mature esterase EstLiu sequence encodes a protein of 273 amino acids residues, with a predicted molecular weight of 30KDa and containing the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243. Although, EstLiu showed 64% similarity with the hypothetical esterase from Chryseobacterium sp. StRB126 (WP_045498424), phylogenetic analysis showed it had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family. The purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). EstLiu showed remarkable activity (75%) at 0°Cand the optimal activity at pH 8.0 and 30°C with good thermostability and quickened inactivation above 60°C. EstLiu retained 81, 103, 67 and 78% of its original activity at 50% (v/v) in ethanol, isopropanol, DMSO and ethylene glycol, respectively. In the presence of Tween 20, Tween 80 and Triton X-100, EstLiu showed 88, 100 and 117% of relative activity. It is also co-factor independent. The high activity at low temperature and desirable stability in organic solvents and salts of this novel family esterase represents a good evidence of novel biocatalyst. Overall, this novel enzyme showed better activity than previously reported esterases in extreme reaction conditions and could promote the reaction in both aqueous and non-aqueous conditions, indicating its great potential for industrial applications.

  7. A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution.

    PubMed

    Lülsdorf, Nina; Vojcic, Ljubica; Hellmuth, Hendrik; Weber, Thomas T; Mußmann, Nina; Martinez, Ronny; Schwaneberg, Ulrich

    2015-06-01

    Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g., p-nitrophenyl acetate) or umbelliferyl-esters are commonly used in directed esterase evolution campaigns. Ongoing challenges in directed esterase evolution are screening formats which offer a broad substrate spectrum, especially for complex aromatic substrates. In this report, a novel continuous high throughput screening system for indirect monitoring of esterolytic activity was developed and validated by detection of phenols employing phenyl benzoate as substrate and p-nitrobenzyl esterase (pNBEBL from Bacillus licheniformis) as catalyst. The released phenol directly reacts with 4-aminoantipyrine yielding the red compound 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2-phenyl-1,2-dihydro-pyrazol-3-one. In this continuous B. licheniformis esterase activity detection system (cBLE-4AAP), the product formation is followed through an increase in absorbance at 509 nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to standard deviation (5 %), linear detection range (15 to 250 μM), lower detection limit (15 μM), and pH (7.4 to 10.4). The cBLE-4AAP screening system was validated by screening a random epPCR pNBEBL mutagenesis library (2000 clones) for improved esterase activity at elevated temperatures. Finally, the variant T3 (Ser378Pro) was identified which nearly retains its specific activity at room temperature (WT 1036 U/mg and T3 929 U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4 °C; WT 170 U/mg to T3 804 U/mg).

  8. Cloning, Characterization, Controlled Overexpression, and Inactivation of the Major Tributyrin Esterase Gene of Lactococcus lactis

    PubMed Central

    Fernández, Leonides; Beerthuyzen, Marke M.; Brown, Julie; Siezen, Roland J.; Coolbear, Tim; Holland, Ross; Kuipers, Oscar P.

    2000-01-01

    The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway. PMID:10742212

  9. Contribution of soil esterase to biodegradation of aliphatic polyester agricultural mulch film in cultivated soils.

    PubMed

    Yamamoto-Tamura, Kimiko; Hiradate, Syuntaro; Watanabe, Takashi; Koitabashi, Motoo; Sameshima-Yamashita, Yuka; Yarimizu, Tohru; Kitamoto, Hiroko

    2015-01-01

    The relationship between degradation speed of soil-buried biodegradable polyester film in a farmland and the characteristics of the predominant polyester-degrading soil microorganisms and enzymes were investigated to determine the BP-degrading ability of cultivated soils through characterization of the basal microbial activities and their transition in soils during BP film degradation. Degradation of poly(butylene succinate-co-adipate) (PBSA) film was evaluated in soil samples from different cultivated fields in Japan for 4 weeks. Both the degradation speed of the PBSA film and the esterase activity were found to be correlated with the ratio of colonies that produced clear zone on fungal minimum medium-agarose plate with emulsified PBSA to the total number colonies counted. Time-dependent change in viable counts of the PBSA-degrading fungi and esterase activities were monitored in soils where buried films showed the most and the least degree of degradation. During the degradation of PBSA film, the viable counts of the PBSA-degrading fungi and the esterase activities in soils, which adhered to the PBSA film, increased with time. The soil, where the film was degraded the fastest, recorded large PBSA-degrading fungal population and showed high esterase activity compared with the other soil samples throughout the incubation period. Meanwhile, esterase activity and viable counts of PBSA-degrading fungi were found to be stable in soils without PBSA film. These results suggest that the higher the distribution ratio of native PBSA-degrading fungi in the soil, the faster the film degradation is. This could be due to the rapid accumulation of secreted esterases in these soils.

  10. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  11. Intracoronary Acetylcholine Provocation Testing for Assessment of Coronary Vasomotor Disorders.

    PubMed

    Ong, Peter; Athanasiadis, Anastasios; Sechtem, Udo

    2016-08-18

    Intracoronary acetylcholine provocation testing (ACH-test) is an established method for assessment of epicardial coronary artery spasm in the catheterization laboratory which was introduced more than 30 years ago. Due to the short half-life of acetylcholine it can only be applied directly into the coronary arteries. Several studies have demonstrated the safety and clinical usefulness of this test. However, acetylcholine testing is only rarely applied in the U.S. or Europe. Nevertheless, it has been shown that 62% of Caucasian patients with stable angina and unobstructed coronary arteries on coronary angiography suffer from coronary vasomotor disorders that can be diagnosed with acetylcholine testing. In recent years it has been appreciated that the ACH-test not only assesses the presence of epicardial spasm but that it can also be useful for the detection of coronary microvascular spam. In such cases no epicardial spasm is seen after injection of acetylcholine but ischemic ECG shifts are present together with a reproduction of the patient's symptoms during the test. This article describes the experience with the ACH-test and its implementation in daily clinical routine.

  12. Acetylcholine causes rooting in leaf explants of in vitro raised tomato (Lycopersicon esculentum Miller) seedlings.

    PubMed

    Bamel, Kiran; Gupta, Shrish Chandra; Gupta, Rajendra

    2007-05-30

    The animal neurotransmitter acetylcholine (ACh) induces rooting and promotes secondary root formation in leaf explants of tomato (Lycopersicon esculentum Miller var. Pusa Ruby), cultured in vitro on Murashige and Skoog's medium. The roots originate from the midrib of leaf explants and resemble taproot. ACh at 10(-5) M was found to be the optimum over a wide range of effective concentrations between 10(-7) and 10(-3) M. The breakdown products, choline and acetate were ineffective even at 10(-3) M concentration. ACh appears to have a natural role in tomato rhizogenesis because exogenous application of neostigmine, an inhibitor of ACh hydrolysis, could mimic the effect of ACh. Neostigmine, if applied in combination with ACh, potentiated the ACh effect.

  13. Endocannabinoids Mediate Muscarinic Acetylcholine Receptor-Dependent Long-Term Depression in the Adult Medial Prefrontal Cortex

    PubMed Central

    Martin, Henry G. S.; Bernabeu, Axel; Lassalle, Olivier; Bouille, Clément; Beurrier, Corinne; Pelissier-Alicot, Anne-Laure; Manzoni, Olivier J.

    2015-01-01

    Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory. Muscarinic acetylcholine receptors (mAChR) in the PFC can induce synaptic plasticity, but the underlying mechanisms remain either opaque or unresolved. We have characterized a form of mAChR mediated long-term depression (LTD) at glutamatergic synapses of layer 5 principal neurons in the adult medial PFC. This mAChR LTD is induced with the mAChR agonist carbachol and inhibited by selective M1 mAChR antagonists. In contrast to other cortical regions, we find that this M1 mAChR mediated LTD is coupled to endogenous cannabinoid (eCB) signaling. Inhibition of the principal eCB CB1 receptor blocked carbachol induced LTD in both rats and mice. Furthermore, when challenged with a sub-threshold carbachol application, LTD was induced in slices pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184, suggesting that the eCB 2-arachidonylglyerol (2-AG) mediates M1 mAChR LTD. Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD. However coupling patterned optical and electrical stimulation to generate local synaptic signaling allowed the reliable induction of LTD. The light—electrical pairing induced LTD was M1 mAChR and CB1 receptor mediated. This shows for the first time that connecting excitatory synaptic activity with coincident endogenously released acetylcholine controls synaptic gain via eCB signaling. Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling. PMID:26648844

  14. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

    PubMed

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

    2017-01-18

    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

  15. A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalosporin C, or acetylated xylan as substrate.

    PubMed

    Martínez-Martínez, Irene; Montoro-García, Silvia; Lozada-Ramírez, José Daniel; Sánchez-Ferrer, Alvaro; García-Carmona, Francisco

    2007-10-15

    A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures.

  16. Cocaine esterase: interactions with cocaine and immune responses in mice.

    PubMed

    Ko, Mei-Chuan; Bowen, Luvina D; Narasimhan, Diwahar; Berlin, Aaron A; Lukacs, Nicholas W; Sunahara, Roger K; Cooper, Ziva D; Woods, James H

    2007-02-01

    Cocaine esterase (CocE) is the most efficient protein catalyst for the hydrolysis of cocaine characterized to date. The aim of this study was to investigate the in vivo potency of CocE in blocking cocaine-induced toxicity in the mouse and to assess CocE's potential immunogenicity. Cocaine toxicity was quantified by measuring the occurrence of convulsions and lethality. Intravenous administration of CocE (0.1-1 mg) 1 min before cocaine administration produced dose-dependent rightward shifts of the dose-response curve for cocaine toxicity. More important, i.v. CocE (0.1-1 mg), given 1 min after the occurrence of cocaine-induced convulsions, shortened the recovery time after the convulsions and saved the mice from subsequent death. Effects of repeated exposures to CocE were evaluated by measuring anti-CocE antibody titers and the protective effects of i.v. CocE (0.32 mg) against toxicity elicited by i.p. cocaine (320 mg/kg) (i.e., 0-17% occurrence of convulsions and lethality). CocE retained its potency against cocaine toxicity in mice after a single prior CocE exposure (0.1-1 mg), and these mice did not show an immune response. CocE retained similar effectiveness in mice after three prior CocE exposures (0.1-1 mg/week for 3 weeks), although these mice displayed 10-fold higher antibody titers. CocE partially lost effectiveness (i.e., 33-50% occurrence of convulsions and lethality) in mice with four prior exposures to CocE (0.1-1 mg/2 week for four times), and these mice displayed approximately 100-fold higher antibody titers. These results suggest that CocE produces robust protection and reversal of cocaine toxicity, indicating CocE's therapeutic potential for acute cocaine toxicity. Repeated CocE exposures may increase its immunogenicity and partially reduce its protective ability.

  17. Inhibitors of Acetylcholinesterase and Butyrylcholinesterase Meet Immunity

    PubMed Central

    Pohanka, Miroslav

    2014-01-01

    Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimer’s disease and other dementias. More recent use is for myasthenia gravis. Many of these inhibitors interact with the second known cholinesterase, butyrylcholinesterase (BChE). Further, evidence shows that acetylcholine plays a role in suppression of cytokine release through a “cholinergic anti-inflammatory pathway” which raises questions about the role of these inhibitors in the immune system. This review covers research and discussion of the role of the inhibitors in modulating the immune response using as examples the commonly available drugs, donepezil, galantamine, huperzine, neostigmine and pyridostigmine. Major attention is given to the cholinergic anti-inflammatory pathway, a well-described link between the central nervous system and terminal effector cells in the immune system. PMID:24893223

  18. Acetylcholine enhancement in the nucleus accumbens prevents addictive behaviors of cocaine and morphine.

    PubMed

    Hikida, Takatoshi; Kitabatake, Yasuji; Pastan, Ira; Nakanishi, Shigetada

    2003-05-13

    Drug addiction poses serious social, medical, and economic problems, but effective treatments for drug addiction are still limited. Cocaine and morphine elevate dopamine levels in the nucleus accumbens (NAc), and the overwhelming actions of dopamine are implicated in reinforcement and addiction of abusive drugs. In our previous studies, we reported the regulatory role of acetylcholine (ACh) in the NAc function by selectively ablating the NAc cholinergic neurons with use of immunotoxin-mediated cell targeting. These studies indicated that ACh and dopamine acted convergently but oppositely on the NAc circuit and that cholinergic cell ablation enhanced long-lasting behavioral changes of cocaine addiction. In this investigation, we showed that immunotoxin-mediated ablation of the NAc cholinergic neurons enhanced not only the sensitivity to morphine in conditioned place preference but also negative reinforcement of morphine withdrawal in conditioned place aversion. Remarkably, acetylcholinesterase (AChE) inhibitors that act on the brain AChE suppressed both cocaine- and morphine-induced conditioned place preference and blocked the induction and persistence of cocaine-evoked hyperlocomotion. Importantly, this inhibition was abolished by ablation of the NAc cholinergic neurons. These results demonstrate that centrally active AChE inhibitors prevent long-lasting behavioral abnormalities associated with cocaine and morphine addictions by potentiating the actions of ACh released from the NAc cholinergic neurons. Centrally active AChE inhibitors could thus be approached as novel and potential therapeutic agents for drug addiction.

  19. A cluster of at least three esterase genes in Lucilia cuprina includes malathion carboxylesterase and two other esterases implicated in resistance to organophosphates

    SciTech Connect

    Smyth, K.A. |; Russell, R.J.; Oakeshott, J.G.

    1994-12-01

    Three distinct malathion carboxylesterase (MCE) phenotypes have been identified among strains of Lucilia cuprina. The high-activity phenotype shows 1.6- and 3.3-fold more MCE specific activity than the intermediate- and low-activity phenotypes, respectively. Flies with high MCE activity are 1000-fold more resistant to malathion than flies with either low or intermediate MCE phenotypes, which are equally susceptible. High and low MCE specific activity are allelic and encoded by the Rmal gene on chromosome 4. Rmal is clustered within one map unit of two other esterase genes, Rop1 and E9, which are implicated in resistance to other organophosphate insecticides. Intermediate MCE specific activity is also inherited within the cluster, although its allelism to Rmal, Rop1, or E9 is unclear. The cluster does not contain the gene for the hemolymph esterase E4, which maps 6.1 map units from Rop1, on the other side of the bubbled wing marker. The cluster appears to be homologous to part of a tandem array of 11 esterase genes on chromosome 3R of Drosophila melanogaster. 41 refs., 4 figs., 2 tabs.

  20. Insecticidal and acetylcholine esterase inhibition activity of Asteraceae plant essential oils and their constituents against adults of the German cockroach (Blattella germanica).

    PubMed

    Yeom, Hwa-Jeong; Jung, Chan-Sik; Kang, Jaesoon; Kim, Junheon; Lee, Jae-Hyeon; Kim, Dong-Soo; Kim, Hyun-Seok; Park, Pil-Sun; Kang, Kyu-Suk; Park, Il-Kwon

    2015-03-04

    The fumigant and contact toxicities of 16 Asteraceae plant essential oils and their constituents against adult male and female Blattella germanica were examined. In a fumigant toxicity test, tarragon oil exhibited 100% and 90% fumigant toxicity against adult male German cockroaches at 5 and 2.5 mg/filter paper, respectively. Fumigant toxicities of Artemisia arborescens and santolina oils against adult male German cockroaches were 100% at 20 mg/filter paper, but were reduced to 60% and 22.5% at 10 mg/filter paper, respectively. In contact toxicity tests, tarragon and santolina oils showed potent insecticidal activity against adult male German cockroaches. Components of active oils were analyzed using gas chromatography, gas chromatography-mass spectrometry, or nuclear magnetic resonance spectrometer. Among the identified compounds from active essential oils, estragole demonstrated potent fumigant and contact toxicity against adult German cockroaches. β-Phellandrene exhibited inhibition of male and female German cockroach acetylcholinesterase activity with IC50 values of 0.30 and 0.28 mg/mL, respectively.

  1. Acetylcholine elongates neuronal growth cone filopodia via activation of nicotinic acetylcholine receptors.

    PubMed

    Zhong, Lei Ray; Estes, Stephen; Artinian, Liana; Rehder, Vincent

    2013-07-01

    In addition to acting as a classical neurotransmitter in synaptic transmission, acetylcholine (ACh) has been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how ACh acts on growth cones to affect growth cone filopodia, structures known to be important for neuronal pathfinding. We addressed this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis in cell culture. ACh treatment caused pronounced filopodial elongation within minutes, an effect that required calcium influx and resulted in the elevation of the intracellular calcium concentration ([Ca]i ). Whole-cell patch clamp recordings showed that ACh caused a reduction in input resistance, a depolarization of the membrane potential, and an increase in firing frequency in B5 neurons. These effects were mediated via the activation of nicotinic acetylcholine receptors (nAChRs), as the nAChR agonist dimethylphenylpiperazinium (DMPP) mimicked the effects of ACh on filopodial elongation, [Ca]i elevation, and changes in electrical activity. Moreover, the nAChR antagonist tubucurarine blocked all DMPP-induced effects. Lastly, ACh acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to ACh by filopodial elongation with a similar time course as growth cones that remained connected to their parent neuron. Our data revealed a critical role for ACh as a modulator of growth cone filopodial dynamics. ACh signaling was mediated via nAChRs and resulted in Ca influx, which, in turn, caused filopodial elongation.

  2. Bacterial cinnamoyl esterase activity screening for the production of a novel functional food product.

    PubMed

    Guglielmetti, Simone; De Noni, Ivano; Caracciolo, Federica; Molinari, Francesco; Parini, Carlo; Mora, Diego

    2008-02-01

    Lactobacillus helveticus MIMLh5 was selected for its strong cinnamoyl esterase activity on chlorogenic acid and employed for the preparation of a food product containing a high concentration of free caffeic acid. The novel food product was demonstrated to display high total antioxidant power and potential probiotic properties.

  3. Bacterial Cinnamoyl Esterase Activity Screening for the Production of a Novel Functional Food Product▿ †

    PubMed Central

    Guglielmetti, Simone; De Noni, Ivano; Caracciolo, Federica; Molinari, Francesco; Parini, Carlo; Mora, Diego

    2008-01-01

    Lactobacillus helveticus MIMLh5 was selected for its strong cinnamoyl esterase activity on chlorogenic acid and employed for the preparation of a food product containing a high concentration of free caffeic acid. The novel food product was demonstrated to display high total antioxidant power and potential probiotic properties. PMID:18165367

  4. Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the present study was to gain new insights into the evolution, homologous recombination and selection pressures imposed on the porcine torovirus (PToV), by examining changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerge...

  5. Purification and characterization of novel extracellular cholesterol esterase from Acinetobacter sp.

    PubMed

    Du, Liangjun; Huo, Ying; Ge, Fanglan; Yu, Jiajun; Li, Wei; Cheng, Guiying; Yong, Bin; Zeng, Lihuang; Huang, Min

    2010-12-01

    CHE4-1, a bacterial strain that belongs to the genus Acinetobacter and expresses high level of inducible extracellular cholesterol esterase (CHE), was isolated from feces of carnivore Panthera pardus var. The cholesterol esterase of the strain CHE4-1 was purified by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography, and then by Sephadex G-50 gel filtration. Different from other known microbial cholesterol esterase, the purified CHE from CHE4-1 strain is a monomer with molecular weight of 6.5 kD and has high activity to both long-chain and short-chain cholesterol ester. Enzymatic activity was enhanced in the presence of metal ion Ca(2+), Zn(2+) and boracic acid, and was not significantly affected by several detergents including sodium cholate, Triton X100 and Tween-80. The enzyme was found to be stable during long-term aqueous storage at 4 °C, indicating its potential as a clinical diagnostic reagent. To the best of our knowledge, this is the first report regarding purification and characterization of CHE from Acinetobacter sp. The results demonstrated that this particular CHE is a novel cholesterol esterase.

  6. Electrochemical biosensor for carbofuran pesticide based on esterases from Eupenicillium shearii FREI-39 endophytic fungus.

    PubMed

    Grawe, Gregory Ferreira; de Oliveira, Tássia Regina; de Andrade Narciso, Esther; Moccelini, Sally Katiuce; Terezo, Ailton José; Soares, Marcos Antonio; Castilho, Marilza

    2015-01-15

    In this work, a biosensor was constructed by physical adsorption of the isolated endophytic fungus Eupenicillium shearii FREI-39 esterase on halloysite, using graphite powder, multi-walled carbon nanotubes and mineral oil for the determination of carbofuran pesticide by inhibition of the esterase using square-wave voltammetry (SWV). Specific esterase activities were determined each 2 days over a period of 15 days of growth in four different inoculation media. The highest specific activity was found on 6th day, with 33.08 U on PDA broth. The best performance of the proposed biosensor was obtained using 0.5 U esterase activity. The carbofuran concentration response was linear in the range from 5.0 to 100.0 µg L(-1) (r=0.9986) with detection and quantification limits of 1.69 µg L(-1) and 5.13 µg L(-1), respectively. A recovery study of carbofuran in spiked water samples showed values ranging from 103.8±6.7% to 106.7±9.7%. The biosensor showed good repeatability and reproducibility and remained stable for a period of 20 weeks. The determination of carbofuran in spiked water samples using the proposed biosensor was satisfactory when compared to the chromatographic reference method. The results showed no significant difference at the 95% confidence level with t-test statistics. The application of enzymes from endophytic fungi in constructing biosensors broadens the biotechnological importance of these microorganisms.

  7. Crystallization and preliminary X-ray diffraction studies of the pneumococcal teichoic acid phosphorylcholine esterase Pce

    SciTech Connect

    Lagartera, Laura; González, Ana; Stelter, Meike; García, Pedro; Kahn, Richard; Menéndez, Margarita; Hermoso, Juan A.

    2005-02-01

    The modular choline-binding protein Pce, the phosphorylcholine esterase from S. pneumoniae, has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a derivative with a gadolinium complex has been collected to 2.7 Å resolution.

  8. Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to phylogenetically classify diverse strains of A. pullulans and determine their production of feruloyl esterase. Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were as...

  9. Cholesterol esterase inhibitory activity of bioactives from leaves of Mangifera indica L

    PubMed Central

    Gururaja, G. M.; Mundkinajeddu, Deepak; Dethe, Shekhar M.; Sangli, Gopala K.; Abhilash, K.; Agarwal, Amit

    2015-01-01

    Background: In the earlier studies, methanolic extract of Mangifera indica L leaf was exhibited hypocholesterol activity. However, the bioactive compounds responsible for the same are not reported so far. Objective: To isolate the bioactive compounds with hypocholesterol activity from the leaf extract using cholesterol esterase inhibition assay which can be used for the standardization of extract. Materials and Methods: The leaf methanolic extract of M. indica (Sindoora variety) was partitioned with ethyl acetate and chromatographed on silica gel to yield twelve fractions and the activity was monitored by using cholesterol esterase inhibition assay. Active fractions were re-chromatographed to yield individual compounds. Results and Discussion: A major compound mangiferin present in the extract was screened along with other varieties of mango leaves for cholesterol esterase inhibition assay. However, the result indicates that compounds other than mangiferin may be active in the extract. Invitro pancreatic cholesterol esterase inhibition assay was used for bioactivity guided fractionation (BAGF) to yield bioactive compound for standardization of extract. Bioactivity guided fractionation afford the active fraction containing 3b-taraxerol with an IC50 value of 0.86μg/ml. Conclusion: This study demonstrates that M. indica methanol extract of leaf have significant hypocholesterol activity which is standardized with 3b-taraxerol, a standardized extract for hypocholesterol activity resulted in development of dietary supplement from leaves of Mangifera indica. PMID:26692750

  10. Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Aspergillus niger ferulic acid esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interactio...

  11. Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

    PubMed Central

    Ishida, K.; Alviano, D.S.; Silva, B.G.; Guerra, C.R.; Costa, A.S.; Nucci, M.; Alviano, C.S.; Rozental, S.

    2012-01-01

    Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes. PMID:22415116

  12. Chaperone-like activities of {alpha}-synuclein: {alpha}-Synuclein assists enzyme activities of esterases

    SciTech Connect

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo . E-mail: ywryu@ajou.ac.kr; Doohun Kim, T. . E-mail: doohunkim@ajou.ac.kr

    2006-08-11

    {alpha}-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of {alpha}-synuclein has not yet been known. Here we have shown that {alpha}-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of {alpha}-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with {alpha}-synuclein. Our results indicate that {alpha}-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.

  13. An Inserted α/β Subdomain Shapes the Catalytic Pocket of Lactobacillus johnsonii Cinnamoyl Esterase

    PubMed Central

    Vu, Clara; Xu, Xiaohui; Cui, Hong; Molloy, Sara; Savchenko, Alexei; Yakunin, Alexander; Gonzalez, Claudio F.

    2011-01-01

    Background Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2. Methodology/Principal Findings We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser106, His225, and Asp197, while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank. Conclusions The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases. PMID:21876742

  14. Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

    PubMed Central

    Martínez-Martínez, Mónica; Alcaide, María; Tchigvintsev, Anatoli; Reva, Oleg; Polaina, Julio; Bargiela, Rafael; Guazzaroni, María-Eugenia; Chicote, Álvaro; Canet, Albert; Valero, Francisco; Rico Eguizabal, Eugenio; Guerrero, María del Carmen; Yakunin, Alexander F.

    2013-01-01

    The esterases and lipases from the α/β hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46′N, 2°59′W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated. PMID:23542620

  15. Crystal Structure and Functional Characterization of an Esterase (EaEST) from Exiguobacterium antarcticum

    PubMed Central

    Kim, Boo-Young; Yoo, Wanki; Ryu, Bum Han; Kim, Han-Woo; Shin, Seung Chul; Kim, Sunghwan; Park, Hyun; Kim, T. Doohun; Lee, Jun Hyuck

    2017-01-01

    A novel microbial esterase, EaEST, from a psychrophilic bacterium Exiguobacterium antarcticum B7, was identified and characterized. To our knowledge, this is the first report describing structural analysis and biochemical characterization of an esterase isolated from the genus Exiguobacterium. Crystal structure of EaEST, determined at a resolution of 1.9 Å, showed that the enzyme has a canonical α/β hydrolase fold with an α-helical cap domain and a catalytic triad consisting of Ser96, Asp220, and His248. Interestingly, the active site of the structure of EaEST is occupied by a peracetate molecule, which is the product of perhydrolysis of acetate. This result suggests that EaEST may have perhydrolase activity. The activity assay showed that EaEST has significant perhydrolase and esterase activity with respect to short-chain p-nitrophenyl esters (≤C8), naphthyl derivatives, phenyl acetate, and glyceryl tributyrate. However, the S96A single mutant had low esterase and perhydrolase activity. Moreover, the L27A mutant showed low levels of protein expression and solubility as well as preference for different substrates. On conducting an enantioselectivity analysis using R- and S-methyl-3-hydroxy-2-methylpropionate, a preference for R-enantiomers was observed. Surprisingly, immobilized EaEST was found to not only retain 200% of its initial activity after incubation for 1 h at 80°C, but also retained more than 60% of its initial activity after 20 cycles of reutilization. This research will serve as basis for future engineering of this esterase for biotechnological and industrial applications. PMID:28125606

  16. Enzyme-linked DNA dendrimer nanosensors for acetylcholine

    NASA Astrophysics Data System (ADS)

    Walsh, Ryan; Morales, Jennifer M.; Skipwith, Christopher G.; Ruckh, Timothy T.; Clark, Heather A.

    2015-10-01

    It is currently difficult to measure small dynamics of molecules in the brain with high spatial and temporal resolution while connecting them to the bigger picture of brain function. A step towards understanding the underlying neural networks of the brain is the ability to sense discrete changes of acetylcholine within a synapse. Here we show an efficient method for generating acetylcholine-detecting nanosensors based on DNA dendrimer scaffolds that incorporate butyrylcholinesterase and fluorescein in a nanoscale arrangement. These nanosensors are selective for acetylcholine and reversibly respond to levels of acetylcholine in the neurophysiological range. This DNA dendrimer architecture has the potential to overcome current obstacles to sensing in the synaptic environment, including the nanoscale size constraints of the synapse and the ability to quantify the spatio-temporal fluctuations of neurotransmitter release. By combining the control of nanosensor architecture with the strategic placement of fluorescent reporters and enzymes, this novel nanosensor platform can facilitate the development of new selective imaging tools for neuroscience.

  17. Changes in Acetylcholine Extracellular Levels during Cognitive Processes

    ERIC Educational Resources Information Center

    Pepeu, Giancarlo; Giovannini, Maria Grazia

    2004-01-01

    Measuring the changes in neurotransmitter extracellular levels in discrete brain areas is considered a tool for identifying the neuronal systems involved in specific behavioral responses or cognitive processes. Acetylcholine (ACh) is the first neurotransmitter whose diffusion from the central nervous system was investigated and whose extracellular…

  18. Clitoria ternatea root extract enhances acetylcholine content in rat hippocampus.

    PubMed

    Rai, K S; Murthy, K D; Karanth, K S; Nalini, K; Rao, M S; Srinivasan, K K

    2002-12-01

    Treatment with 100 mg/kg of Clitoria ternatea aqueous root extract (CTR), for 30 days in neonatal and young adult age groups of rat, significantly increased acetylcholine (ACh) content in their hippocampi as compared to age matched controls. Increase in ACh content in their hippocampus may be the neurochemical basis for their improved learning and memory.

  19. Mechanisms of Action of Anticholinesterases and Oximes on Acetylcholine Receptors

    DTIC Science & Technology

    1988-07-23

    J.F. and D.B. Sanders. The management of patients with myasthenia gravis , in Myasthenia Gravis (E.X. Albuquerque and A.T. Eldefrawi, eds.), Chapman...Eldefrawi. Affinity of myasthenia drugs to acetylcholinesterase and acetylcholine receptor. Biochem. Med. 10:258-265 (1974). 9. Carpenter, D.O., L.A

  20. Binding of quinolizidine alkaloids to nicotinic and muscarinic acetylcholine receptors.

    PubMed

    Schmeller, T; Sauerwein, M; Sporer, F; Wink, M; Müller, W E

    1994-09-01

    Fourteen quinolizidine alkaloids, isolated from Lupinus albus, L. mutabilis, and Anagyris foetida, were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. Of the compounds tested, the alpha-pyridones, N-methylcytisine and cytisine, showed the highest affinities at the nicotinic receptor, while several quinolizidine alkaloid types were especially active at the muscarinic receptor.

  1. Acetylcholine receptors in the retinas of the α7 nicotinic acetylcholine receptor knockout mouse

    PubMed Central

    Souza, Fred G. Oliveira; Bruce, Kady S.; Strang, Christianne E.; Morley, Barbara J.; Keyser, Kent T.

    2014-01-01

    Purpose The α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer’s disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse. Methods To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes. Results In the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype

  2. Polyester with Pendent Acetylcholine-Mimicking Functionalities Promotes Neurite Growth.

    PubMed

    Wang, Shaofei; Jeffries, Eric; Gao, Jin; Sun, Lijie; You, Zhengwei; Wang, Yadong

    2016-04-20

    Successful regeneration of nerves can benefit from biomaterials that provide a supportive biochemical and mechanical environment while also degrading with controlled inflammation and minimal scar formation. Herein, we report a neuroactive polymer functionalized by covalent attachment of the neurotransmitter acetylcholine (Ach). The polymer was readily synthesized in two steps from poly(sebacoyl diglyceride) (PSeD), which previously demonstrated biocompatibility and biodegradation in vivo. Distinct from prior acetylcholine-biomimetic polymers, PSeD-Ach contains both quaternary ammonium and free acetyl moieties, closely resembling native acetylcholine structure. The polymer structure was confirmed via (1)H nuclear magnetic resonance and Fourier-transform infrared spectroscopy. Hydrophilicity, charge, and thermal properties of PSeD-Ach were determined by tensiometer, zetasizer, differential scanning calorimetry, and thermal gravimetric analysis, respectively. PC12 cells exhibited the greatest proliferation and neurite outgrowth on PSeD-Ach and laminin substrates, with no significant difference between these groups. PSeD-Ach yielded much longer neurite outgrowth than the control polymer containing ammonium but no the acetyl group, confirming the importance of the entire acetylcholine-like moiety. Furthermore, PSeD-Ach supports adhesion of primary rat dorsal root ganglions and subsequent neurite sprouting and extension. The sprouting rate is comparable to the best conditions from previous report. Our findings are significant in that they were obtained with acetylcholine-like functionalities in 100% repeating units, a condition shown to yield significant toxicity in prior publications. Moreover, PSeD-Ach exhibited favorable mechanical and degradation properties for nerve tissue engineering application. Humidified PSeD-Ach had an elastic modulus of 76.9 kPa, close to native neural tissue, and could well recover from cyclic dynamic compression. PSeD-Ach showed a gradual in

  3. Differential inhibition of nicotine- and acetylcholine-evoked currents through alpha4beta2 neuronal nicotinic receptors by tobacco cembranoids in Xenopus oocytes.

    PubMed

    Eaton, Misty J; Ospina, Claudia A; Rodríguez, Abimael D; Eterović, Vesna A

    2004-08-05

    In tobacco, there are two types of compounds that interact with neuronal nicotinic acetylcholine receptors (nnAChRs) in the brain. The first is the addictive component of tobacco and an agonist of these receptors, nicotine. The second are cyclic diterpenoids called cembranoids that non-competitively inhibit many types of nnAChRs. Nictotinic receptors composed of alpha4beta2 subunits are the predominant type of nicotinic receptors in the brain. These alpha4beta2 receptors are up-regulated upon chronic exposure to nicotine and have been implicated in nicotine addiction. The present study was designed to determine whether the inhibitory effects of two cembranoids from tobacco [(1S, 2E, 4R, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (4R) and its diastereoisomer (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (4S)] were comparable on acetylcholine (ACh) and nicotine-evoked currents through alpha4beta2 nnAChRs. alpha4beta2 nnAChRs from rat brain were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The dose-response curves for acetylcholine and nicotine were hyperbolic and bell-shaped, respectively. Although there was no difference in the potency between cembranoids 4R and 4S, both of these cembranoids more potently inhibited nicotine-induced currents than acetylcholine-induced currents. Furthermore, both cembranoids were more potent inhibitors of this receptor when they were preincubated for 1 min prior to application of agonist. The finding that cembranoids preferentially inhibit nicotine-induced currents over those elicited by the natural neurotransmitter acetylcholine may have important implications when developing strategies to prevent nicotine addiction and tobacco use.

  4. Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

    PubMed

    Ma, W; Maric, D; Li, B S; Hu, Q; Andreadis, J D; Grant, G M; Liu, Q Y; Shaffer, K M; Chang, Y H; Zhang, L; Pancrazio, J J; Pant, H C; Stenger, D A; Barker, J L

    2000-04-01

    Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

  5. Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases.

    PubMed

    Kim, Jinyeong; Kim, Seungbum; Yoon, Sangyoung; Hong, Eunsoo; Ryu, Yeonwoo

    2015-08-01

    Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = -16.2 ± 0.2 and E = 0.7 ± 0.0): V138G (ee% = 35.9 ± 1.0 and E = 3.0 ± 0.1) and V138G/L200R (ee% = 89.2 ± 0.2 and E = 19.5 ± 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.

  6. The effects of postnatal alcohol exposure and galantamine on the context pre-exposure facilitation effect and acetylcholine efflux using in vivo microdialysis.

    PubMed

    Perkins, Amy E; Fadel, Jim R; Kelly, Sandra J

    2015-05-01

    Fetal alcohol spectrum disorders (FASD) are characterized by damage to multiple brain regions, including the hippocampus, which is involved in learning and memory. The acetylcholine neurotransmitter system provides major input to the hippocampus and is a possible target of developmental alcohol exposure. Alcohol (3.0 g/kg/day) was administered via intubation to male rat pups (postnatal day [PD] 2-10; ethanol-treated [ET]). Controls received a sham intubation (IC) or no treatment (NC). Acetylcholine efflux was measured using in vivo microdialysis (PD 32-35). ET animals were not different at baseline, but had decreased K(+)/Ca(2+)-induced acetylcholine efflux compared to NC animals and an enhanced acetylcholine response to galantamine (acetylcholinesterase inhibitor; 2.0 mg/kg) compared to both control groups. A separate cohort of animals was tested in the context pre-exposure facilitation effect task (CPFE; PD 30-32) following postnatal alcohol exposure and administration of galantamine (2.0 mg/kg; PD 11-30). Neither chronic galantamine nor postnatal alcohol exposure influenced performance in the CPFE task. Using immunohistochemistry, we found that neither alcohol exposure nor behavioral testing significantly altered the density of vesicular acetylcholine transporter or alpha7 nicotinic acetylcholine receptor in the ventral hippocampus (CA1). In the medial septum, the average number of choline acetyltransferase (ChAT+) cells was increased in ET animals that displayed the context-shock association; there were no changes in IC and NC animals that learned the context-shock association or in any animals that were in the control task that entailed no learning. Taken together, these results indicate that the hippocampal acetylcholine system is significantly disrupted under conditions of pharmacological manipulations (e.g., galantamine) in alcohol-exposed animals. Furthermore, ChAT was up‑regulated in ET animals that learned the CPFE, which may account for their ability

  7. Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats

    SciTech Connect

    Ray, A.; Liu, J.; Karanth, S.; Gao, Y.; Brimijoin, S.; Pope, C.

    2009-05-01

    into enzyme-inhibitor interactions. Furthermore, the proliferation/migration of cells containing high amounts of cholinesterase just adjacent to a dialysis probe could affect the recovery and thus detection of extracellular acetylcholine in microdialysis studies.

  8. Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine.

    PubMed

    Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M; DeSimone, John A; Lyall, Vijay

    2015-01-01

    Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.

  9. Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine

    PubMed Central

    Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M.; DeSimone, John A.; Lyall, Vijay

    2015-01-01

    Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol. PMID:26039516

  10. Biocatalytic Resolution of Rac-α-Ethyl-2-Oxo-Pyrrolidineacetic Acid Methyl Ester by Immobilized Recombinant Bacillus cereus Esterase.

    PubMed

    Zheng, Jian-Yong; Liu, Yin-Yan; Luo, Wei-Feng; Zheng, Ren-Chao; Ying, Xiang-Xian; Wang, Zhao

    2016-04-01

    A new esterase-producing strain (Bacillus cereus WZZ001) which exhibiting high hydrolytic activity and excellent enantioselectivity on rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester (R, S-1) has been isolated from soil sample by our laboratory. In this study, the stereoselective hydrolysis of (R, S-1) was performed using the recombinant Bacillus cereus esterase which expressed in Escherichia coli BL21 (DE3). Under the optimized conditions of pH 8.0, 35 °C, and concentration of substrate 400 mM, a successful enzymatic resolution was achieved with an e.e. s of 99.5 % and conversion of 49 %. Immobilization considerably increased the reusability of the recombinant esterase; the immobilized enzyme showed excellent reusability during 6 cycles of repeated 2 h reactions at 35 °C. Thereby, it makes the recombinant B. cereus esterase a usable biocatalyst for industrial application.

  11. Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus.

    PubMed

    Sugiyama, Kou-ichi; Tezuka, Takafumi

    2011-10-01

    Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint.

  12. [Effect of acetylcholine and acetylcholinesterase on the activity of contractile vacuole of Amoeba proteus].

    PubMed

    Bagrov, Ia Iu; Manusova, N B

    2011-01-01

    Acetylcholine (ACh, 1 microM) stimulates activity of the contractile vacuole of proteus. The effect of ACh is not mimicked by its analogs which are not hydrolyzed by acetylcholinesterase (AChE), i. e., carbacholine and 5-methylfurmethide. The effect of ACh is not sensitive to the blocking action of M-cholinolytics, atropine and mytolone, but is suppressed by N-cholinolytic, tubocurarine. The inhibitors of AChE, eserine (0.01 microM) and armine (0.1 microM), suppress the effect of ACh on amoeba contractile vacuole. ACh does not affect activation of contractile vacuole induced by arginine-vasopressin (1 microM), but it blocks such effect of opiate receptors agonist, dynorphin A1-13 (0.01 microM). This effect of ACh is also suppressed by the inhibitors of AChE. These results suggest that, in the above-described effects of ACh, AChE acts not as an antagonist, but rather as a synergist.

  13. Comparative study of human intestinal and hepatic esterases as related to enzymatic properties and hydrolizing activity for ester-type drugs.

    PubMed

    Inoue, M; Morikawa, M; Tsuboi, M; Ito, Y; Sugiura, M

    1980-08-01

    In attempts to determine the exact role of intestinal esterase in the body, we purified esterases from human intestinal mucosa and liver, and compared the enzymatic properties and substrate specificities with those of purified esterases. Esterase from human liver was purified 58-fold, by treatment with butanol, DE-52 and DEAE Sephadex A-50 column chromatographies, Sephadex G-200 gel filtration, and isoelectric focusing. The purified preparation showed a single band by polyacylamide gel electrophoresis. The molecular weights of intestinal and hepatic esterases were determined to be 53,000-55,000 and 180,000, respectively, by gel filtration on Sephadex G-200. The activity of the purified intestinal and hepatic esterases was strongly inhibited by diethyl-p-nitrophenyl phosphate and diisopropyl fluorophosphate, and was not inhibited by eserine sulfate and p-chloromercuribenzoate. Moreover, the purified esterases hydrolyzed ester-type drugs such as aspirin, clofibrate, indanyl carbenicillin and procaine. Hepatic esterase had properties similar to those of intestinal esterase with respect to the sensitivity to organophosphate and the substrate specificity. However, the two purified esterases differed in properties such as molecular weight, isoelectric point, thermostability and optimal pH.

  14. The dual-acting H3 receptor antagonist and AChE inhibitor UW-MD-71 dose-dependently enhances memory retrieval and reverses dizocilpine-induced memory impairment in rats.

    PubMed

    Khan, Nadia; Saad, Ali; Nurulain, Syed M; Darras, Fouad H; Decker, Michael; Sadek, Bassem

    2016-01-15

    Both the histamine H3 receptor (H3R) and acetylcholine esterase (AChE) are involved in the regulation of release and metabolism of acetylcholine and several other central neurotransmitters. Therefore, dual-active H3R antagonists and AChE inhibitors (AChEIs) have shown in several studies to hold promise to treat cognitive disorders like Alzheimer's disease (AD). The novel dual-acting H3R antagonist and AChEI 7-(3-(piperidin-1-yl)propoxy)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline (UW-MD-71) with excellent selectivity profiles over both the three other HRs as well as the AChE's isoenzyme butyrylcholinesterase (BChE) shows high and balanced in vitro affinities at both H3R and AChE with IC50 of 33.9nM and hH3R antagonism with Ki of 76.2nM, respectively. In the present study, the effects of UW-MD-71 (1.25-5mg/kg, i.p.) on acquisition, consolidation, and retrieval in a one-trial inhibitory avoidance task in male rats were investigated applying donepezil (DOZ) and pitolisant (PIT) as reference drugs. Furthermore, the effects of UW-MD-71 on memory deficits induced by the non-competitive N-methyl-d-aspartate (NMDA) antagonist dizocilpine (DIZ) were tested. Our results indicate that administration of UW-MD-71 before the test session dose-dependently increased performance and enhanced procognitive effect on retrieval. However neither pre- nor post-training acute systemic administration of UW-MD-71 facilitated acquisition or consolidation. More importantly, UW-MD-71 (2.5mg/kg, i.p.) ameliorated the DIZ-induced amnesic effects. Furthermore, the procognitive activity of UW-MD-71 in retrieval was completely reversed and partly abrogated in DIZ-induced amnesia when rats were pretreated with the centrally-acting H2R antagonist zolantidine (ZOL), but not with the CNS penetrant H1R antagonist pyrilamine (PYR). These results demonstrate the procognitive effects of UW-MD-71 in two in vivo memory models, and are to our knowledge the first demonstration in vivo that a potent dual

  15. Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes.

    PubMed

    Rodríguez, María Cecilia; Loaces, Inés; Amarelle, Vanesa; Senatore, Daniella; Iriarte, Andrés; Fabiano, Elena; Noya, Francisco

    2015-01-01

    A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40 °C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.

  16. Acute promyelocytic leukemia, hypogranular variant, with uncharacteristic staining with chloroacetate esterase.

    PubMed

    Dunphy, C H; Polski, J M; Johns, G; Evans, H L; Gardner, L J

    2001-06-01

    A diagnosis of the hypogranular variant of acute promyelocytic leukemia (APLv) may be difficult to establish based on cytomorphology alone. However, the great majority of cases have a classical immunophenotype by flow cytometric immunophenotyping (FCI) (CD13+, CD33+, dim CD64+, HLA-DR-, and CD34-) and a classical enzyme cytochemical (EC) staining pattern. [intensely staining with myeloperoxidase, Sudan Black B, and chloroacetate esterase (CAE) and negative with alpha'-naphthyl acetate and butyrate esterases]. Although the immunophenotype of APLv by FCI has varied in the literature (HLA-DR +/- and CD34 +/-), the EC staining pattern has remained constant. We report a case of APLv with characteristic cytomorphology, compatible FCI data (CD13+, CD33+, dim CD64+, HLA-DR +/-, and CD34-), chromosomal detection of t(15; 17), and molecular detection of the PML/RAR alpha fusion gene; however, staining of the leukemic cells with CAE was quite uncharacteristic. We describe our findings.

  17. Heterozygosity of the sheep: Polymorphism of 'malic enzyme', isocitrate dehydrogenase (NADP+), catalase and esterase.

    PubMed

    Baker, C M; Manwell, C

    1977-04-01

    In contrast to other reports, it is found that the sheep has approximately as much enzyme variation as man. Most of the genetically interpretable enzyme variation in heart, liver, kidney and muscle from 52 sheep (Merinos or Merino crosses) is in the NADP-dependent dehydrogenases [two 'malic enzymes' and the supernatant isocitrate dehydrogenase (NADP+)] and in the esterases. Ten different loci for NAD-dependent dehydrogenases are electrophoretically monomorphic, as are five different NADH diaphorases from heart muscle and 15 different major proteins from skeletal muscle. It is highly statistically significant that NADP-dependent dehydrogenases and esterases are polymorphic but representatives of several other major classes of enzymes are not. The physiological significance of this polymorphism may be related to the role of these enzymes in growth and detoxication, sheep having been selected by man for faster growth, of wool or of carcass, and for grazing a wide variety of plants.

  18. Improved biomass degradation using fungal glucuronoyl-esterases-hydrolysis of natural corn fiber substrate.

    PubMed

    d'Errico, Clotilde; Börjesson, Johan; Ding, Hanshu; Krogh, Kristian B R M; Spodsberg, Nikolaj; Madsen, Robert; Monrad, Rune Nygaard

    2016-02-10

    Lignin-carbohydrate complexes (LCCs) are in part responsible for the recalcitrance of lignocellulosics in relation to industrial utilization of biomass for biofuels. Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 have been proposed to be able to degrade ester LCCs between glucuronic acids in xylans and lignin alcohols. By means of synthesized complex LCC model substrates we provide kinetic data suggesting a preference of fungal GEs for esters of bulky arylalkyl alcohols such as ester LCCs. Furthermore, using natural corn fiber substrate we report the first examples of improved degradation of lignocellulosic biomass by the use of GEs. Improved C5 sugar, glucose and glucuronic acid release was observed when heat pretreated corn fiber was incubated in the presence of GEs from Cerrena unicolor and Trichoderma reesei on top of different commercial cellulase/hemicellulase preparations. These results emphasize the potential of GEs for delignification of biomass thereby improving the overall yield of fermentable sugars for biofuel production.

  19. Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate.

    PubMed

    Morono, Yuki; Takano, Suguru; Miyanaga, Kazuhiko; Tanji, Yasunori; Unno, Hajime; Hori, Katsutoshi

    2004-03-01

    Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

  20. Pseudomonas aeruginosa biofilm growth inhibition on medical plastic materials by immobilized esterases and acylase.

    PubMed

    Kisch, Johannes Martin; Utpatel, Christian; Hilterhaus, Lutz; Streit, Wolfgang R; Liese, Andreas

    2014-09-05

    Biofilms are matrix-encapsulated cell aggregates that cause problems in technical and health-related areas; for example, 65 % of all human infections are biofilm associated. This is mainly due to their ameliorated resistance against antimicrobials and immune systems. Pseudomonas aeruginosa, a biofilm-forming organism, is commonly responsible for nosocomial infections. Biofilm development is partly mediated by signal molecules, such as acyl-homoserine lactones (AHLs) in Gram-negative bacteria. We applied horse liver esterase, porcine kidney acylase, and porcine liver esterase; these can hydrolyze AHLs, thereby inhibiting biofilm formation. As biofilm infections are often related to foreign material introduced into the human body, we immobilized the enzymes on medical plastic materials. Biofilm formation was quantified by Crystal Violet staining and confocal laser scanning microscopy, revealing up to 97 % (on silicone), 54 % (on polyvinyl chloride), and 77 % (on polyurethane) reduced biomass after 68 h growth.

  1. Is Esterase-P Encoded by a Cryptic Pseudogene in Drosophila Melanogaster?

    PubMed Central

    Balakirev, E. S.; Ayala, F. J.

    1996-01-01

    We have amplified and sequenced the gene encoding Esterase-P (Est-P) in 10 strains of Drosophila melanogaster. Three premature termination codons occur in the coding region of the gene in two strains. This observation, together with other indirect evidence, leads us to propose that Est-P may be a pseudogene in D. melanogaster. Est-P would be a ``cryptic'' pseudogene, in the sense that it retains intact the coding sequence (without stop codons and other alterations usually observed in pseudogenes) in most D. melanogaster strains. We conjecture that the β-esterase cluster may consist in other Drosophila species of functional and nonfunctional genes. We also conjecture that the rarity of detected pseudogenes in Drosophila may be due to the difficulty of discovering them, because most of them are cryptic. PMID:8978040

  2. Release of short chain fatty acids from cream lipids by commercial lipases and esterases.

    PubMed

    Saerens, K; Descamps, D; Dewettinck, K

    2008-02-01

    Lipases and esterases are frequently used in dairy production processes to enhance the buttery flavour of the end product. Short chain fatty acids, and especially butanoic acid, play a key role in this and different enzymes with specificity towards short chain fatty acids are commercially available as potent flavouring tools. We have compared six lipases/esterases associated with buttery flavour production. Although specificity to short chain fatty acids was ascribed to each enzyme, clear differences in free fatty acid profiles were found when these enzymes were applied on cream. Candida cylindraceae lipase was the most useful enzyme for buttery flavour production in cream with the highest yield of free fatty acids (57 g oleic acid 100 g(-1) fat), no release of long chain fatty acids and specificity towards butanoic acid.

  3. Esterase-Sensitive Prodrugs with Tunable Release Rates and Direct Generation of Hydrogen Sulfide.

    PubMed

    Zheng, Yueqin; Yu, Bingchen; Ji, Kaili; Pan, Zhixiang; Chittavong, Vayou; Wang, Binghe

    2016-03-24

    Prodrugs that release hydrogen sulfide upon esterase-mediated cleavage of an ester group followed by lactonization are described herein. By modifying the ester group and thus its susceptibility to esterase, and structural features critical to the lactonization rate, H2 S release rates can be tuned. Such prodrugs directly release hydrogen sulfide without the involvement of perthiol species, which are commonly encountered with existing H2 S donors. Additionally, such prodrugs can easily be conjugated to another non-steroidal anti-inflammatory agent, leading to easy synthesis of hybrid prodrugs. As a biological validation of the H2 S prodrugs, the anti-inflammatory effects of one such prodrug were examined by studying its ability to inhibit LPS-induced TNF-α production in RAW 264.7 cells. This type of H2 S prodrugs shows great potential as both research tools and therapeutic agents.

  4. Intrinsic temperature sensitivity of influenza C virus hemagglutinin-esterase-fusion protein.

    PubMed

    Takashita, Emi; Muraki, Yasushi; Sugawara, Kanetsu; Asao, Hironobu; Nishimura, Hidekazu; Suzuki, Koji; Tsuji, Takashi; Hongo, Seiji; Ohara, Yoshiro; Ohara, Yoshihiro; Kawaoka, Yoshihiro; Ozawa, Makoto; Matsuzaki, Yoko

    2012-12-01

    Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.

  5. Total cholesterol, high density lipoprotein cholesterol and choline esterase in overseas and Japanese university students.

    PubMed

    Nakamura, S

    1985-04-01

    Serum lipids were studied in 97 overseas and 282 Japanese university students. As compared with Japanese, serum total cholesterol levels were low and high density lipoprotein/total cholesterol ratio was high in the overseas students, especially in Chinese and Korean students. 30-39-year-old Chinese students, moreover, showed elevated high density lipoprotein levels. Choline esterase levels were significantly lower in 30-39-year-old Chinese and Korean students than in Japanese and Taiwanese.

  6. [Methods of increasing the activity of extracellular esterase, beta-fructofuranosidase and proteases of wine yeast].

    PubMed

    Abdurazakova, S Kh; Salomov, Kh T

    1975-01-01

    Upon regular fermentation changes in the activity of the enzymes esterase, beta-fructofuranosidase and protease of the yeast Saccharomyces mini of the Parkent I race were examined. The maximum activity of the enzymes occurred in the stationary phase of the yeast growth. An increase in the activity of the above enzymes was shown possible during a prolonged stabilization of the stationary conditions in the process of a continuous chemostat cultivation of wine yeast.

  7. A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

    PubMed

    Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G

    2015-03-01

    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.

  8. Esterase Activated Carbonyl Sulfide/Hydrogen Sulfide (H2S) Donors.

    PubMed

    Chauhan, Preeti; Bora, Prerona; Ravikumar, Govindan; Jos, Swetha; Chakrapani, Harinath

    2017-01-06

    Hydrogen sulfide (H2S) is a mediator of a number of cellular processes, and modulating cellular levels of this gas has emerged as an important therapeutic area. Localized generation of H2S is thus very useful but highly challenging. Here, we report pivaloyloxymethyl-based carbonothioates and carbamothioates that are activated by the enzyme, esterase, to generate carbonyl sulfide (COS), which is hydrolyzed to H2S.

  9. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst)

    PubMed Central

    Martins, Julia M.; DeMarco, Ricardo; Jameson, David M.; Castro, Aline M.; Bossolan, Nelma R. S.; Wallace, B. A.; Araujo, Ana P. U.

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required. PMID:27351338

  10. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

    PubMed

    Lopes, Jose L S; Yoneda, Juliana S; Martins, Julia M; DeMarco, Ricardo; Jameson, David M; Castro, Aline M; Bossolan, Nelma R S; Wallace, B A; Araujo, Ana P U

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

  11. Characterization of patatin esterase activity in AOT-isooctane reverse micelles.

    PubMed

    Jiménez, M; Escribano, J; Gandía-Herrero, F; Chazarra, S; Cabanes, J; García-Carmona, F; Pérez-Gilabert, M

    2002-01-01

    Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported not only to serve as a storage protein but also to exhibit lipid acyl hydrolase (LAH) activity. In this study patatin is characterized in AOT-isooctane reverse micelles. The influence on the enzymatic activity of characteristic parameters of reverse micelles, w(o) (= H(2)O/AOT), and the percentage of H(2)O, theta, were investigated. The results obtained show that patatin esterase activity varies with w(o) but remains constant throughout the range of theta values studied. The variation with w(o) showed that the activity follows an S-shaped behavior pattern, reaching a maximum at about w(o) = 20 for 2% H(2)O. Patatin esterase activity was compared with p-nitrophenyl (PNP) fatty acid esters of different chain lengths. The activity was much higher for PNP-caprylate. The pH optimum was 6.0, different from the value obtained when patatin esterase activity was measured in mixed micelle systems. The optimal temperature was 35 degrees C, above which the activity decreased to almost zero. The kinetic parameters were also evaluated (K(m) = 10 mM, V(m) = 158 microM/min, V(m)/K(m) = 15.8 x 10(-3) min(-1)). This paper shows the suitability of reverse micelles for measuring patatin esterase activity, since it allows the study of the enzyme in similar conditions to that prevailing in vivo.

  12. Characterisation of a cold adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

    PubMed

    Dong, Juan; Gasmalla, Mohammed A A; Zhao, Wei; Sun, Jingtao; Liu, Wenyu; Wang, Mingming; Han, Liang; Yang, Ruijin

    2016-07-13

    A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No.1, Tianshan, China, and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room-temperature plasma (ARTP) method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30°C, pH 9.0 and 25°C, pH 8.5, respectively. EstTB11 was thermally more stable (50°C for 1 hour) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0°C and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4°C. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry. This article is protected by copyright. All rights reserved.

  13. A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

    PubMed Central

    Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

    2014-01-01

    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

  14. Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome

    PubMed Central

    De Santi, Concetta; Willassen, Nils Peder

    2016-01-01

    Background The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. Results MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes. PMID:27433797

  15. Purification of neuropathy target esterase from avian brain after prelabelling with [3H]diisopropyl phosphorofluoridate.

    PubMed

    Rüffer-Turner, M E; Read, D J; Johnson, M K

    1992-01-01

    Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.

  16. Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex

    PubMed Central

    Beiranvand, F; Zlabinger, C; Orr-Urtreger, A; Ristl, R; Huck, S; Scholze, P

    2014-01-01

    Background and purpose Nicotinic acetylcholine receptors (nACh receptors) play a central role in the habenulo-interpeduncular system. We studied nicotine-induced release of NA and ACh in the habenula and interpeduncular nucleus (IPN). Experimental approach The habenula and IPN were loaded with [3H]-choline or [3H]-NA and placed in superfusion chambers. [3H]-ACh release was also stimulated using nicotinic agonists, electrical pulses and elevated [KCl]o in hippocampal and cortical slices from rats, wild-type mice and mice lacking α5, α7, β2, or β4 nACh receptor subunits. Finally, we analysed nACh receptor subtypes in the IPN using immunoprecipitation. Key results Nicotine induced release of [3H]-ACh in the IPN of rats and mice. This release was calcium-dependent but not blocked by tetrodotoxin (TTX); moreover, [3H]-ACh release was abolished in β4-knockout mice but was unaffected in β2- and α5-knockout mice. In contrast, nicotine-induced release of [3H]-NA in the IPN and habenula was blocked by TTX and reduced in both β2-knockout and β4-knockout mice, and dose–response curves were right-shifted in α5-knockout mice. Although electrical stimuli triggered the release of both transmitters, [3H]-ACh release required more pulses delivered at a higher frequency. Conclusions and implications Our results confirm previous findings that β4-containing nACh receptors are critical for [3H]-ACh release in the mouse IPN. Experiments using α5-knockout mice also revealed that unlike in the hippocampus, nicotine-induced [3H]-NA release in the habenulo-interpeduncular system is altered in this knockout model. As α5-containing nACh receptors play a key role in nicotine intake, our results add NA to the list of transmitters involved in this mechanism. PMID:25041479

  17. A Dynamical Role for Acetylcholine in Synaptic Renormalization

    PubMed Central

    Fink, Christian G.; Murphy, Geoffrey G.; Zochowski, Michal; Booth, Victoria

    2013-01-01

    Although sleep is a fundamental behavior observed in virtually all animal species, its functions remain unclear. One leading proposal, known as the synaptic renormalization hypothesis, suggests that sleep is necessary to counteract a global strengthening of synapses that occurs during wakefulness. Evidence for sleep-dependent synaptic downscaling (or synaptic renormalization) has been observed experimentally, but the physiological mechanisms which generate this phenomenon are unknown. In this study, we propose that changes in neuronal membrane excitability induced by acetylcholine may provide a dynamical mechanism for both wake-dependent synaptic upscaling and sleep-dependent downscaling. We show in silico that cholinergically-induced changes in network firing patterns alter overall network synaptic potentiation when synaptic strengths evolve through spike-timing dependent plasticity mechanisms. Specifically, network synaptic potentiation increases dramatically with high cholinergic concentration and decreases dramatically with low levels of acetylcholine. We demonstrate that this phenomenon is robust across variation of many different network parameters. PMID:23516342

  18. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Tochitsky, Ivan; Banghart, Matthew R.; Mourot, Alexandre; Yao, Jennifer Z.; Gaub, Benjamin; Kramer, Richard H.; Trauner, Dirk

    2012-02-01

    Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

  19. Spin labeled acetylcholine analogs: studies of cholinergic receptor.

    PubMed

    Rosen, G M; Abou-Donia, M B; Yeh, J Z; Menzel, D B

    1975-10-01

    Some spin-labeled acetylcholine analogs, in which the number of methylene groups between the quaternary nitrogen and the ether oxygen ranged between 1-5, were synthesized to study drug interacitons with acetylcholine receptors. None of the compounds tested, with the exception of the one that contained 2 methylene groups (SL-2) had any cholinergic activity. SL-2 was not capable of producing any nicotinic cholinomimetic activity. On the other hand it proved to have a very weak nicotinic cholinolytic activity on the receptors of the frog satorius muscle. This compound exhibited strong antagonism against muscarinic receptors of the isolated frog heart. The muscarinic cholinolytic action of the spin-label ACh analog is discussed in terms of the molecular perturbation theory of drug action.

  20. Structural role of a conserved active site cis proline in the Thermotoga maritima acetyl esterase from the carbohydrate esterase family 7.

    PubMed

    Singh, Mrityunjay K; Manoj, Narayanan

    2017-04-01

    A conserved cis proline residue located in the active site of Thermotoga maritima acetyl esterase (TmAcE) from the carbohydrate esterase family 7 (CE7) has been substituted by alanine. The residue was known to play a crucial role in determining the catalytic properties of the enzyme. To elucidate the structural role of the residue, the crystal structure of the Pro228Ala variant (TmAcEP228A ) was determined at 2.1 Å resolution. The replacement does not affect the overall secondary, tertiary, and quaternary structures and moderately decreases the thermal stability. However, the wild type cis conformation of the 227-228 peptide bond adopts a trans conformation in the variant. Other conformational changes in the tertiary structure are restricted to residues 222-226, preceding this peptide bond and are located away from the active site. Overall, the results suggest that the conserved proline residue is responsible for the cis conformation of the peptide and shapes the geometry of the active site. Elimination of the pyrrolidine ring results in the loss of van der Waals and hydrophobic interactions with both the alcohol and acyl moeities of the ester substrate, leading to significant impairment of the activity and perturbation of substrate specificity. Furthermore, a cis-to-trans conformational change arising out of residue changes at this position may be associated with the evolution of divergent activity, specificity, and stability properties of members constituting the CE7 family. Proteins 2017; 85:694-708. © 2016 Wiley Periodicals, Inc.

  1. Production and characterization of a tributyrin esterase from Lactobacillus plantarum suitable for cheese lipolysis.

    PubMed

    Esteban-Torres, M; Mancheño, J M; de las Rivas, B; Muñoz, R

    2014-11-01

    Lactobacillus plantarum is a lactic acid bacterium that can be found during cheese ripening. Lipolysis of milk triacylglycerols to free fatty acids during cheese ripening has fundamental consequences on cheese flavor. In the present study, the gene lp_1760, encoding a putative esterase or lipase, was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_1760 protein was biochemically characterized. Lp_1760 hydrolyzed p-nitrophenyl esters of fatty acids from C2 to C16, with a preference for p-nitrophenyl butyrate. On triglycerides, Lp_1760 showed higher activity on tributyrin than on triacetin. Although optimal conditions for activity were 45°C and pH 7, Lp_1760 retains activity under conditions commonly found during cheese making and ripening. The Lp_1760 showed more than 50% activity at 5°C and exhibited thermal stability at high temperatures. Enzymatic activity was strongly inhibited by sodium dodecyl sulfate and phenylmethylsulfonyl fluoride. The Lp_1760 tributyrin esterase showed high activity in the presence of NaCl, lactic acid, and calcium chloride. The results suggest that Lp_1760 might be a useful tributyrin esterase to be used in cheese manufacturing.

  2. Newly Identified Thermostable Esterase from Sulfobacillus acidophilus: Properties and Performance in Phthalate Ester Degradation

    PubMed Central

    Zhang, Xiao-Yan; Fan, Xiang; Qiu, Yong-Jun; Li, Cheng-Yuan; Xing, Shuai; Zheng, Yi-Tao

    2014-01-01

    EstS1, a newly identified thermostable esterase from Sulfobacillus acidophilus DSM10332, was heterologously expressed in Escherichia coli and shown to enzymatically degrade phthalate esters (PAEs) to their corresponding monoalkyl PAEs. The optimal pH and temperature of the esterase were found to be 8.0 and 70°C, respectively. The half-life of EstS1 at 60°C was 15 h, indicating that the enzyme had good thermostability. The specificity constant (kcat/Km) of the enzyme for p-nitrophenyl butyrate was as high as 6,770 mM−1 s−1. The potential value of EstS1 was demonstrated by its ability to effectively hydrolyze 35 to 82% of PAEs (10 mM) within 2 min at 37°C, with all substrates being completely degraded within 24 h. At 60°C, the time required for complete hydrolysis of most PAEs was reduced by half. To our knowledge, this enzyme is a new esterase identified from thermophiles that is able to degrade various PAEs at high temperatures. PMID:25149523

  3. Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.

    PubMed

    Tao, Weixin; Lee, Myung Hwan; Yoon, Mi-Young; Kim, Jin-Cheol; Malhotra, Shweta; Wu, Jing; Hwang, Eul Chul; Lee, Seon-Woo

    2011-12-01

    Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.

  4. Metagenomic mining for thermostable esterolytic enzymes uncovers a new family of bacterial esterases

    PubMed Central

    Zarafeta, Dimitra; Moschidi, Danai; Ladoukakis, Efthymios; Gavrilov, Sergey; Chrysina, Evangelia D.; Chatziioannou, Aristotelis; Kublanov, Ilya; Skretas, Georgios; Kolisis, Fragiskos N.

    2016-01-01

    Biocatalysts exerting activity against ester bonds have a broad range of applications in modern biotechnology. Here, we have identified a new esterolytic enzyme by screening a metagenomic sample collected from a hot spring in Kamchatka, Russia. Biochemical characterization of the new esterase, termed EstDZ2, revealed that it is highly active against medium chain fatty acid esters at temperatures between 25 and 60 °C and at pH values 7–8. The new enzyme is moderately thermostable with a half-life of more than six hours at 60 °C, but exhibits exquisite stability against high concentrations of organic solvents. Phylogenetic analysis indicated that EstDZ2 is likely an Acetothermia enzyme that belongs to a new family of bacterial esterases, for which we propose the index XV. One distinctive feature of this new family, is the presence of a conserved GHSAG catalytic motif. Multiple sequence alignment, coupled with computational modelling of the three-dimensional structure of EstDZ2, revealed that the enzyme lacks the largest part of the “cap” domain, whose extended structure is characteristic for the closely related Family IV esterases. Thus, EstDZ2 appears to be distinct from known related esterolytic enzymes, both in terms of sequence characteristics, as well as in terms of three-dimensional structure. PMID:27991516

  5. Cloning, expression and characterization of a novel esterase from a South China Sea sediment metagenome

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Li, Fuchao; Chen, Huaxin; Zhao, Jin; Yan, Jinfei; Jiang, Peng; Li, Ronggui; Zhu, Baoli

    2015-07-01

    Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. ScsEst01 was successfully co-expressed in Escherichia coli BL21 (DE3) with chaperones (dnaK-dnaJ-grpE) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2+. The enzyme was characterized using p -nitrophenol butyrate as a substrate. ScsEst01 had the highest lipolytic activity at 35°C and pH 8.0, indicative of a meso-thermophilic alkaline esterase. ScsEst01 was thermostable at 20°C. The lipolytic activity of scsEst01 was strongly increased by Fe2+, Mn2+ and 1% Tween 80 or Tween 20.

  6. Structural and functional profile of the carbohydrate esterase gene complement in Phytophthora infestans.

    PubMed

    Ospina-Giraldo, Manuel D; McWalters, Jessica; Seyer, Lauren

    2010-12-01

    The plant cell cuticle is the first obstacle for penetration of the host by plant pathogens. To breach this barrier, most pathogenic fungi employ a complex assortment of cell wall-degrading enzymes including carbohydrate esterases, glycoside hydrolases, and polysaccharide lyases. We characterized the full complement of carbohydrate esterase-coding genes in three Phytophthora species and analyzed the expression of cutinase in vitro and in planta; we also determined the cutinase allele distribution in multiple isolates of P. infestans. Our investigations revealed that there are 49, 21, and 37 esterase homologs in the P. infestans, P. ramorum, and P. sojae genomes, respectively, with a considerable number predicted to be extracellular. Four cutinase gene copies were found in both the P. infestans and P. ramorum genomes, while 16 copies were found in P. sojae. Transcriptional analyses of cutinase in P. infestans revealed that its expression level during infection is significantly upregulated at all time points compared to that of the same gene in mycelium grown in vitro. Expression achieves maximum values at 15 hpi, declining at subsequent time points. These results may suggest, therefore, that cutinase most likely plays a role in P. infestans pathogenicity.

  7. Conversion of a Rhizopus chinensis lipase into an esterase by lid swapping.

    PubMed

    Yu, Xiao-Wei; Zhu, Shan-Shan; Xiao, Rong; Xu, Yan

    2014-06-01

    In an effort to explore the feasibility of converting a lipase into an esterase by modifying the lid region, we designed and characterized two novel Rhizopus chinensis lipase variants by lid swapping. The substrate specificity of an R. chinensis lipase was successfully modified toward water-soluble substrates, that is, turned into an esterase, by replacing the hydrophobic lid with a hydrophilic lid from ferulic acid esterase from Aspergillus niger Meanwhile, as a comparison, the lid of R. chinensis lipase was replaced by a hydrophobic lid from Rhizomucor miehei lipase, which did not alter its substrate specificity but led to a 5.4-fold higher catalytic efficiency (k*cat/K*m) toward p-nitrophenyl laurate. Based on the analysis of structure-function relationships, it suggests that the amphipathic nature of the lid is very important for the substrate specificity. This study provides new insight into the structural basis of lipase specificities and a way to tune the substrate preference of lipases.

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

    PubMed

    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.

  9. Acetylcholine mediates behavioral and neural post-error control.

    PubMed

    Danielmeier, Claudia; Allen, Elena A; Jocham, Gerhard; Onur, Oezguer A; Eichele, Tom; Ullsperger, Markus

    2015-06-01

    Humans often commit errors when they are distracted by irrelevant information and no longer focus on what is relevant to the task at hand. Adjustments following errors are essential for optimizing goal achievement. The posterior medial frontal cortex (pMFC), a key area for monitoring errors, has been shown to trigger such post-error adjustments by modulating activity in visual cortical areas. However, the mechanisms by which pMFC controls sensory cortices are unknown. We provide evidence for a mechanism based on pMFC-induced recruitment of cholinergic projections to task-relevant sensory areas. Using fMRI in healthy volunteers, we found that error-related pMFC activity predicted subsequent adjustments in task-relevant visual brain areas. In particular, following an error, activity increased in those visual cortical areas involved in processing task-relevant stimulus features, whereas activity decreased in areas representing irrelevant, distracting features. Following treatment with the muscarinic acetylcholine receptor antagonist biperiden, activity in visual areas was no longer under control of error-related pMFC activity. This was paralleled by abolished post-error behavioral adjustments under biperiden. Our results reveal a prominent role of acetylcholine in cognitive control that has not been recognized thus far. Regaining optimal performance after errors critically depends on top-down control of perception driven by the pMFC and mediated by acetylcholine. This may explain the lack of adaptivity in conditions with reduced availability of cortical acetylcholine, such as Alzheimer's disease.

  10. Identification of a family of muscarinic acetylcholine receptor genes

    SciTech Connect

    Bonner, T.I.; Buckley, N.J.; Young, A.C.; Brann, M.R.

    1987-07-31

    Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.

  11. Acetylcholine in the rat pituitary: a possible humoral factor.

    PubMed

    Egozi, Y; Kloog, Y; Fleminger, G; Sokolovsky, M

    1988-12-20

    Significant amounts of acetylcholine (ACh) were detected in each of the 3 lobes of the rat pituitary (3-6 pmol/anterior lobe, 3 pmol/intermediate lobe and 1.8 pmol/posterior lobe). In the anterior lobes of cyclic rats the levels of ACh varied with the estrous cycle, with daily peaks being observed on the days of proestrus and estrus. The occurrence of ACh, apparently as a humoral factor, appears to be unique to the anterior pituitary.

  12. A hyperpolarized choline molecular probe for monitoring acetylcholine synthesis.

    PubMed

    Allouche-Arnon, Hyla; Gamliel, Ayelet; Barzilay, Claudia M; Nalbandian, Ruppen; Gomori, J Moshe; Karlsson, Magnus; Lerche, Mathilde H; Katz-Brull, Rachel

    2011-01-01

    Choline as a reporter molecule has been investigated by in vivo magnetic resonance for almost three decades. Accumulation of choline metabolites (mainly the phosphorylated forms) had been observed in malignancy in preclinical models, ex-vivo, in vivo and in patients. The combined choline metabolite signal appears in (1) H-MRS of the brain and its relative intensity had been used as a diagnostic factor in various conditions. The advent of spin hyperpolarization methods for in vivo use has raised interest in the ability to follow the physiological metabolism of choline into acetylcholine in the brain. Here we present a stable-isotope labeled choline analog, [1,1,2,2-D(4) ,2-(13) C]choline chloride, that is suitable for this purpose. In this analog, the (13) C position showed 24% polarization in the liquid state, following DNP hyperpolarization. This nucleus also showed a long T(1) (35 s) at 11.8 T and 25 °C, which is a prerequisite for hyperpolarized studies. The chemical shift of this (13) C position differentiates choline and acetylcholine from each other and from the other water-soluble choline metabolites, namely phosphocholine and betaine. Enzymatic studies using an acetyltransferase enzyme showed the synthesis of the deuterated-acetylcholine form at thermal equilibrium conditions and in a hyperpolarized state. Analysis using a comprehensive model showed that the T(1) of the formed hyperpolarized [1,1,2,2-D(4) ,2-(13) C]acetylcholine was 34 s at 14.1 T and 37 °C. We conclude that [1,1,2,2-D(4) ,2-(13) C]choline chloride is a promising new molecular probe for hyperpolarized metabolic studies and discuss the factors related to its possible use in vivo.

  13. Role of B-esterases in assessing toxicity of organophosphorus (chlorpyrifos, malathion) and carbamate (carbofuran) pesticides to Daphnia magna.

    PubMed

    Barata, Carlos; Solayan, Arun; Porte, Cinta

    2004-02-10

    In this study, the cladoceran Daphnia magna was exposed to two model organophosphorous and one carbamate pesticides including malathion, chlorpyrifos and carbofuran to assess acetylcholinesterase (AChE) and carboxylesterase (CbE) inhibition and recovery patterns and relate those responses with individual level effects. Our results revealed differences in enzyme inhibition and recovery patterns among the studied esterase enzymes and pesticides. CbE was more sensitive to organophosphorous than AChE, whereas both CbE and AChE showed equivalent sensitivities to the carbamate carbofuran. Recovery patterns of AChE and CbE activities following exposure to the studied pesticides were similar with 80-100% recoveries taking place 12 and 96 h after exposure to organophosphorous and carbamates pesticides, respectively. The physiological role of AChE and CbE inhibition patterns in Daphnia was examined by using organophosphorous and carbamate compounds alone and with specific inhibitors of CbE. Under exposure to organophosphorous pesticides, survival of Daphnia juveniles was impaired at AChE inhibition levels higher than 50% whereas under exposure to the carbamate carbofuran low levels of AChE inhibition affected mortality. Inhibition of CbE by 80-90% increased toxicity to organophosphorous and carbamate pesticides by up to two- and four-fold, respectively. Our results suggest that both AChE and CbE enzymes are involved in determining toxicity of Daphnia to the studied chemicals and that AChE inhibition levels higher than 50% can be considered of environmental concern to Daphnia species.

  14. Acetylcholine-induced phosphorylation in isolated outer hair cells.

    PubMed

    Szõnyi, M; Csermely, P; Sziklai, I

    1999-03-01

    Two groups of isolated, surviving outer hair cells (OHCs) of guinea pig cochleas (n = 20, for each group) were treated with 10 microM acetylcholine or acetylcholine plus strichnine (an alpha9 nAChR antagonist), respectively, under short-term tissue culture conditions. The protein content of the cell homogenates was separated by SDS-polyacrylamide gel electrophoresis, Western blotted and labelled with an antibody against phosphoserine residues. Signals were detected using the ECL system. Acetylcholine challenge of the OHCs resulted in a difference in the pattern of phosphorylated proteins from those of strichnine pretreated cells. A 220 kDa and a 120 kDa protein expressed a more intense phosphorylated state in the ACh group compared with the ACh plus strichnine group. The 220 kDa phosphoprotein is in the range of the cytoskeletal protein beta-fodrin, whereas the 120 kDa fraction is similar to alpha-fodrin or an ankyrin isoform. Phosphorylation of proteins due to activation of the AChR by agonist can play a role in the signalling mechanism between receptor activation and increase in the electromotile capability of isolated OHCs.

  15. Nicotinic Acetylcholine Receptor Signaling in Tumor Growth and Metastasis

    PubMed Central

    Singh, Sandeep; Pillai, Smitha; Chellappan, Srikumar

    2011-01-01

    Cigarette smoking is highly correlated with the onset of a variety of human cancers, and continued smoking is known to abrogate the beneficial effects of cancer therapy. While tobacco smoke contains hundreds of molecules that are known carcinogens, nicotine, the main addictive component of tobacco smoke, is not carcinogenic. At the same time, nicotine has been shown to promote cell proliferation, angiogenesis, and epithelial-mesenchymal transition, leading to enhanced tumor growth and metastasis. These effects of nicotine are mediated through the nicotinic acetylcholine receptors that are expressed on a variety of neuronal and nonneuronal cells. Specific signal transduction cascades that emanate from different nAChR subunits or subunit combinations facilitate the proliferative and prosurvival functions of nicotine. Nicotinic acetylcholine receptors appear to stimulate many downstream signaling cascades induced by growth factors and mitogens. It has been suggested that antagonists of nAChR signaling might have antitumor effects and might open new avenues for combating tobacco-related cancer. This paper examines the historical data connecting nicotine tumor progression and the recent efforts to target the nicotinic acetylcholine receptors to combat cancer. PMID:21541211

  16. The intracellular pathway of the acetylcholine-induced contraction in cat detrusor muscle cells

    PubMed Central

    An, J Y; Yun, H S; Lee, Y P; Yang, S J; Shim, J O; Jeong, J H; Shin, C Y; Kim, J H; Kim, D S; Sohn, U D

    2002-01-01

    The present study was aimed to investigate intracellular pathways involved in acetylcholine (ACh)-induced contraction in cat detrusor muscle cells Contraction was expressed as per cent shortening of length of individually isolated smooth muscle cells obtained by enzymatic digestion. Dispersed intact and permeabilized cells were prepared for the treatment of drugs and antibody to enzymes, respectively. Using Western blot, we confirmed the presence of related proteins. The maximal contraction to ACh was generated at 10−11 M. This response was preferentially antagonized by M3 muscarinic receptor antagonist ρ-fluoro-hexahydrosiladifenidol (ρF-HSD) but not by the M1 antagonist pirenzepine and the M2 muscarinic receptor antagonist methoctramine. We identified G-proteins Gq/11, Gs, G0, Gi1, Gi2 and Gi3 in the bladder detrusor muscle. ACh-induced contraction was selectively inhibited by Gq/11 antibody but not to other G subunit. The phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor neomycin reduced ACh-induced contraction. However, the inhibitors of the phospholipase D, the phospholipase A2 and protein kinase C did not attenuate the ACh-induced contraction. ACh-induced contraction was inhibited by antibody to PLC-β1 but not PLC-β3 and PLC-γ. Thapsigargin or strontium, which depletes or blocks intracellular calcium release, inhibited ACh-induced contraction. Inositol 1,4,5-triphosphate (IP3) receptor inhibitor heparin reduced ACh-induced contraction. These results suggest that in cat detrusor muscle contraction induced by ACh is mediated via M3 muscarinic receptor-dependent activation of Gq/11 and PLC-β1 and IP3-dependent Ca2+ release. PMID:12429572

  17. The endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes.

    PubMed

    Oz, Murat; Ravindran, Arippa; Diaz-Ruiz, Oscar; Zhang, Li; Morales, Marisela

    2003-09-01

    The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned alpha7 subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 microM) induced-currents in a concentration-dependent manner (10 nM to 30 microM), with an IC50 value of 229.7 +/- 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca2+ dependent Cl- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca2+-free bathing solution containing 2 mM Ba2+. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) alpha7 receptors. This effect was not mediated by CB1 or CB2 receptors, as neither the selective CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh alpha7 receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and

  18. Evaluation of the sensitivity of the novel α4β2* nicotinic acetylcholine receptor PET radioligand 18F-(−)-NCFHEB to increases in synaptic acetylcholine levels in rhesus monkeys

    PubMed Central

    Gallezot, Jean-Dominique; Esterlis, Irina; Bois, Frederic; Zheng, Ming-Qiang; Lin, Shu-fei; Kloczynski, Tracy; Krystal, John H.; Huang, Yiyun; Sabri, Osama; Carson, Richard E.; Cosgrove, Kelly P.

    2014-01-01

    Objective 18F-(−)-NCFHEB (also known as 18F-(−)-Flubatine) is a new radioligand to image α4β2* nicotinic acetylcholine receptors in vivo with positron emission tomography (PET), with faster kinetics than previous radioligands such as 18F-2-F-A85380. The goal of this study was to assess the sensitivity of 18F-(−)-NCFHEB-PET to increases in synaptic acetylcholine concentration induced by acetylcholinesterase inhibitors. Methods Two rhesus monkeys were scanned four times each on a Focus 220 scanner: first at baseline, then during two bolus plus infusions of physostigmine (0.06–0.28 mg/kg), and finally following a bolus injection of donepezil (0.25 mg/kg). The arterial input function and the plasma free fraction fP were measured. 18F-(−)-NCFHEB volume of distribution VT was estimated using the multilinear analysis MA1 and then normalized by plasma free fraction fP. Results 18F-(−)-NCFHEB fP was 0.89±0.04. At baseline, 18F-(−)-NCFHEB VT/fP ranged from 7.9±1.3 mL plasma/cm3 tissue in the cerebellum to 34.3±8.4 mL plasma/cm3 tissue in the thalamus. Physostigmine induced a dose-dependent reduction of 18F-(−)-NCFHEB VT/fP of 34±9% in the putamen, 32±8% in the thalamus, 25±8% in the cortex, and 23±10% in the hippocampus. With donepezil, 18F-(−)-NCFHEB VT/fP was reduced by 24±2%, 14+3% and 14±5%, 10±6% in the same regions. Conclusion 18F-(−)- NCFHEB can be used to detect changes in synaptic acetylcholine concentration and is a promising tracer to study acetylcholine dynamics with shorter scan durations than previous radioligands. PMID:25043426

  19. Release of acetylcholine by syringin, an active principle of Eleutherococcus senticosus, to raise insulin secretion in Wistar rats.

    PubMed

    Liu, Ko Yu; Wu, Yang-Chang; Liu, I-Min; Yu, Wen Chen; Cheng, Juei-Tang

    2008-03-28

    The present study is designed to screen the effect of syringin, an active principle purified from the rhizome and root parts of Eleutherococcus senticosus (Araliaceae), on the plasma glucose and investigate the possible mechanisms. Plasma glucose decreased in a dose-dependent manner 60 min after intravenous injection of syringin into fasting Wistar rats. In parallel to the decrease of plasma glucose, increases of plasma insulin level as well as the plasma C-peptide was also observed in rats receiving same treatment. Both the plasma glucose lowering action and the raised plasma levels of insulin and C-peptide induced by syringin were also inhibited by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP), the antagonist of the muscarinic M3 receptors, but not affected by the ganglionic nicotinic antagonist, pentolinium or hexamethonium. Moreover, disruption of synaptic available acetylcholine (ACh) using an inhibitor of choline uptake, hemicholinium-3, or vesicular acetylcholine transport, vesamicol, abolished these actions of syringin. Also, physostigmine at concentration sufficient to inhibit acetylcholinesterase enhanced the actions of syringin. Mediation of ACh release from the nerve terminals to enhance insulin secretion by syringin can thus be considered. The results suggest that syringin has an ability to raise the release of ACh from nerve terminals, which in turn to stimulate muscarinic M3 receptors in pancreatic cells and augment the insulin release to result in plasma glucose lowering action.

  20. Comparison of (/sup 3/H)nicotine and (/sup 3/H)acetylcholine binding in mouse brain: regional distribution

    SciTech Connect

    Sershen, H.; Reith, M.E.; Hashim, A.; Lajtha, A.

    1985-06-01

    In a continuing study of nicotine binding sites, the authors determined the relative amount of nicotine binding and acetylcholine binding in various brain regions of C57/BL and of DBA mice. Although midbrain showed the highest and cerebellum the lowest binding for both (/sup 3/H)nicotine and (/sup 3/H)acetylcholine, the ratio of nicotine to acetylcholine binding showed a three-fold regional variation. Acetylcholine inhibition of (/sup 3/H)nicotine binding indicated that a portion of nicotine binding was not inhibited by acetylcholine. These results indicate important differences between the binding of (+/-)-(/sup 3/H)nicotine and that of (/sup 3/H)acetylcholine.

  1. Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries

    PubMed Central

    Goldman, Daniel H.; Aggarwal, Nitin T.; Chawengsub, Yuttana; Falck, J. R.; Campbell, William B.

    2011-01-01

    Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-l-arginine (l-NA; 30 μM) blocked relaxations in the thoracic aorta, and l-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K+ channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [14C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF1α, PGE2, 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11(R),12(S),15(S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western

  2. [Sugammadex. New pharmacological concept for antagonizing rocuronium and vecuronium].

    PubMed

    Sparr, H J; Booij, L H; Fuchs-Buder, T

    2009-01-01

    Up to now only acetylcholine esterase inhibitors, such as neostigmine, were available as antagonists of residual neuromuscular blocks. Sugammadex is a modified gamma-cyclodextrin that binds rocuronium and chemically similar aminosteroidal muscle relaxants, such as vecuronium. The underlying mechanism of action is new and differs completely from that of acetylcholine esterase inhibitors. This review summarizes data published so far within the framework of the licensing procedure about the efficacy, safety and side-effects of sugammadex and presents potential new anesthesiological concepts using this compound.

  3. In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor.

    PubMed

    Ulrich, H; Ippolito, J E; Pagán, O R; Eterović, V A; Hann, R M; Shi, H; Lis, J T; Eldefrawi, M E; Hess, G P

    1998-11-24

    The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.

  4. Genome-wide analysis of esterase-like genes in the striped rice stem borer, Chilo suppressalis.

    PubMed

    Wang, Baoju; Wang, Ying; Zhang, Yang; Han, Ping; Li, Fei; Han, Zhaojun

    2015-06-01

    The striped rice stem borer, Chilo suppressalis, a destructive pest of rice, has developed high levels of resistance to certain insecticides. Esterases are reported to be involved in insecticide resistance in several insects. Therefore, this study systematically analyzed esterase-like genes in C. suppressalis. Fifty-one esterase-like genes were identified in the draft genomic sequences of the species, and 20 cDNA sequences were derived which encoded full- or nearly full-length proteins. The putative esterase proteins derived from these full-length genes are overall highly diversified. However, key residues that are functionally important including the serine residue in the active site are conserved in 18 out of the 20 proteins. Phylogenetic analysis revealed that most of these genes have homologues in other lepidoptera insects. Genes CsuEst6, CsuEst10, CsuEst11, and CsuEst51 were induced by the insecticide triazophos, and genes CsuEst9, CsuEst11, CsuEst14, and CsuEst51 were induced by the insecticide chlorantraniliprole. Our results provide a foundation for future studies of insecticide resistance in C. suppressalis and for comparative research with esterase genes from other insect species.

  5. Biological Activity of sym-Triazines with Acetylcholine-like Substitutions as Multitarget Modulators of Alzheimer’s Disease

    PubMed Central

    2013-01-01

    The bioactivities of two novel compounds (TAE-1 and TAE-2) that contain a sym-triazine scaffold with acetylcholine-like substitutions are examined as promising candidate agents against Alzheimer’s disease. Inhibition of amyloid-β fibril formation in the presence of Aβ1–42, evaluated by Thioflavin T fluorescence, demonstrated comparable or improved activity to a previously reported pentapeptide-based fibrillogenesis inhibitor, iAβ5p. Destabilization of Aβ1–42 assemblies by TAE-1 and TAE-2 was confirmed by scanning electron microscopy imaging. sym-Triazine inhibition of acetylcholinesterase (AChE) activity was observed in cytosol extracted from differentiated human SH-SY5Y neuronal cells and also using human erythrocyte AChE. The sym-triazine derivatives were well tolerated by these cells and promoted beneficial effects on human neurons, upregulating expression of synaptophysin, a synaptic marker protein, and MAP2, a neuronal differentiation marker. PMID:23472585

  6. Cerebral Acetylcholine and Choline Contents and Turnover Following Low-Dose Acetylcholinesterase Inhibitor Treatment in Rats

    DTIC Science & Technology

    2006-05-01

    mesencephalon, neocortex, piriform cortex, and striatum. Ch uptake from blood and ACh turnover were estimated from D4Ch and D4ACh concentrations in...measured by gas-chromatography mass- spectrometry in hippocampus, infundibulum, mesencephalon, neocortex, piriform cortex, and striatum. Ch uptake from...memory (hippocampus, neocortex, and piriform cortex) or autonomic regulation (mesencephalon, infundibulum). MATERIALS AND METHODS Animals

  7. Nicotinic acetylcholine receptors: from basic science to therapeutics.

    PubMed

    Hurst, Raymond; Rollema, Hans; Bertrand, Daniel

    2013-01-01

    Substantial progress in the identification of genes encoding for a large number of proteins responsible for various aspects of neurotransmitter release, postsynaptic detection and downstream signaling, has advanced our understanding of the mechanisms by which neurons communicate and interact. Nicotinic acetylcholine receptors represent a large and well-characterized family of ligand-gated ion channels that is expressed broadly throughout the central and peripheral nervous system, and in non-neuronal cells. With 16 mammalian genes identified that encode for nicotinic receptors and the ability of the subunits to form heteromeric or homomeric receptors, the repertoire of conceivable receptor subtype combinations is enormous and offers unique possibilities for the design and development of new therapeutics that target nicotinic acetylcholine receptors. The aim of this review is to provide the reader with recent insights in nicotinic acetylcholine receptors from genes, structure and function to diseases, and with the latest findings on the pharmacology of these receptors. Although so far only a few nicotinic drugs have been marketed or are in late stage development, much progress has been made in the design of novel chemical entities that are being explored for the treatment of various diseases, including addiction, depression, ADHD, cognitive deficits in schizophrenia and Alzheimer's disease, pain and inflammation. A pharmacological analysis of these compounds, including those that were discontinued, can improve our understanding of the pharmacodynamic and pharmacokinetic requirements for nicotinic 'drug-like' molecules and will reveal if hypotheses on therapies based on targeting specific nicotinic receptor subtypes have been adequately tested in the clinic.

  8. Single acetylcholine receptor channel currents recorded at high hydrostatic pressures.

    PubMed Central

    Heinemann, S H; Stühmer, W; Conti, F

    1987-01-01

    A technique for performing patch-clamp experiments under high hydrostatic (oil) pressure is described. The method allows the transfer of whole cell or membrane patches in a recording configuration into a pressure vessel, where pressure can be increased up to 60 MPa (approximately equal to 600 bar). We have studied in this way the pressure dependence of single acetylcholine receptor channels in excised "outside-out" membrane patches from cultured rat muscle cells. In the range of 0.1 to 60 MPa the open channel conductance in 140 mM NaCl solutions did not vary by more than 2%, which implies that the translocation of sodium ions through the channel pore does not involve steps with significant activation volumes. At high acetylcholine concentrations (20 microM) bursts of single-channel activity allowed measurements of the mean open and mean closed times of the channel. Pressurization to 40 MPa increased both mean open and mean closed times giving apparent activation volumes of about 59 and 139 A3, respectively. This implies a net volume increase of 80 A3, associated with the transition from the agonist-free state to the open state of the channel, which may be partially associated with the agonist-binding step. All the observed pressure effects were reversible. The activation volumes for the gating of acetylcholine receptor channels are comparable to those of sodium and potassium channels in the squid giant axon, suggesting that there is some basic common mechanism in the operation of ion-channel proteins. Images PMID:2437577

  9. Increasing Hippocampal Acetylcholine Levels Enhances Behavioral Performance in an Animal Model of Diencephalic Amnesia

    PubMed Central

    Roland, Jessica J.; Mark, Katherine; Vetreno, Ryan P.; Savage, Lisa M.

    2008-01-01

    Pyrithiamine-induced thiamine deficiency (PTD) was used to produce a rodent model of Wernicke-Korsakoff syndrome that results in acute neurological disturbances, thalamic lesions, and learning and memory impairments. There is also cholinergic septo-hippocampal dysfunction in the PTD model. Systemic (Experiment 1) and intrahippocampal (Experiment 2) injections of the acetylcholinesterase inhibitor physostigmine were administered to determine if increasing acetylcholine levels would eliminate the behavioral impairment produced by PTD. Prior to spontaneous alternation testing, rats received injections of either physostigmine (systemic = 0.075 mg/kg; intrahippocampal = 20, 40 ng/µl) or saline. In Experiment 2, intrahippocampal injections of physostigmine significantly enhanced alternation rates in the PTD-treated rats. In addition, although intrahippocampal infusions of 40 ng of physostigmine increased the available amount of ACh in both Pair-fed (PF) and PTD rats, it did so to a greater extent in PF rats. The increase in ACh levels induced by the direct hippocampal application of physostigmine in the PTD model likely increased activation of the extended limbic system, which was dysfunctional, and therefore led to recovery of function on the spontaneous alternation task. In contrast, the lack of behavioral improvement by intrahippocampal physostigmine infusion in the PF rats, despite a greater rise in hippocampal ACh levels, supports the theory that there is a optimal range of cholinergic tone for optimal behavioral and hippocampal function. PMID:18706897

  10. Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

    PubMed

    Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

    2016-05-01

    Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity.

  11. Structure-activity relationships of benzylidene anabaseines in nicotinic acetylcholine receptors of cockroach nerve cords.

    PubMed

    Sultana, Israt; Ikeda, Izumi; Ozoe, Yoshihisa

    2002-09-01

    Ten analogues of 6'-chloro-3-benzylideneanabaseine (CBA) bearing substituents at the ortho- and the para-positions of the phenyl group were synthesized, together with two related compounds. The affinity of the synthesized compounds for nicotinic acetylcholine receptors (nAChRs) in the nerve cord of the American cockroach (Periplaneta americana L.) was examined by the radioligand binding assay using [(3)H]epibatidine (EPI), a nAChR agonist. All 12 tested compounds inhibited [(3)H]EPI binding, showing K(i) values ranging from 14.6 to 6830nM. The potency variation of para-substituted CBA analogues was explained by the steric (Delta B(1)) and electronic (sigma(p)) parameters of the para-substituents, or by the steric parameter and the charge of the N1 nitrogen atom (qN(1)). Among the CBA analogues, only two compounds containing a dimethylamino group and a methoxy group at the para-position showed high insecticidal activity against the German cockroach (Blattella germanica) when injected after pretreatment with metabolic inhibitors. High-affinity analogues of CBA might be suitable probes for use in classifying and characterizing insect nAChR subtypes.

  12. Acetylcholine elevation relieves cognitive rigidity and social deficiency in a mouse model of autism.

    PubMed

    Karvat, Golan; Kimchi, Tali

    2014-03-01

    Autism spectrum disorders (ASD) are defined by behavioral deficits in social interaction and communication, repetitive stereotyped behaviors, and restricted interests/cognitive rigidity. Recent studies in humans and animal-models suggest that dysfunction of the cholinergic system may underlie autism-related behavioral symptoms. Here we tested the hypothesis that augmentation of acetylcholine (ACh) in the synaptic cleft by inhibiting acetylcholinesterase may ameliorate autistic phenotypes. We first administered the acetylcholinesterase inhibitor (AChEI) Donepezil systemically by intraperitoneal (i.p.) injections. Second, the drug was injected directly into the rodent homolog of the caudate nucleus, the dorsomedial striatum (DMS), of the inbred mouse strain BTBR T+tf/J (BTBR), a commonly-used model presenting all core autism-related phenotypes and expressing low brain ACh levels. We found that i.p. injection of AChEI to BTBR mice significantly relieved autism-relevant phenotypes, including decreasing cognitive rigidity, improving social preference, and enhancing social interaction, in a dose-dependent manner. Microinjection of the drug directly into the DMS, but not into the ventromedial striatum, led to significant amelioration of the cognitive-rigidity and social-deficiency phenotypes. Taken together, these findings provide evidence of the key role of the cholinergic system and the DMS in the etiology of ASD, and suggest that elevated cognitive flexibility may result in enhanced social attention. The potential therapeutic effect of AChEIs in ASD patients is discussed.

  13. Agarwood induced laxative effects via acetylcholine receptors on loperamide-induced constipation in mice.

    PubMed

    Kakino, Mamoru; Izuta, Hiroshi; Ito, Tetsuro; Tsuruma, Kazuhiro; Araki, Yoko; Shimazawa, Masamitsu; Oyama, Masayoshi; Iinuma, Munekazu; Hara, Hideaki

    2010-01-01

    Agarwood (Aquilaria sinensis, Aquilaria crasna) is well known as an incense in the oriental region such as Thailand, Taiwan, and Cambodia, and is used as a digestive in traditional medicine. We investigated the laxative effects and mechanism of agarwood leaves extracted with ethanol (EEA-1, Aquilaria sinensis; EEA-2, Aquilaria crasna). EEA-1, EEA-2, the main constituents of EEAs (mangiferin, and genkwanin-5-O-primeveroside), and senna increased the frequency and weight of stools in loperamide-induced constipation model mice. EEA-1 and EEA-2 did not induce diarrhea as a side effect, but senna induced severe diarrhea. EEA-1 and senna increased gastro-intestinal (GI) transit in the model mice. EEA-1, but not senna, also increased the intestinal tension of isolated jejunum and ileum in guinea pigs, and the tension increase was blocked by atropine, a muscarinic receptor antagonist, but not by other inhibitors (granicetron, pyrilamine, or bradykinin-antagonist peptide). Furthermore, the increase in frequency and weight of stools induced by EEA-1 were blocked by pre-administration of atropine in the model mice. These findings indicate that EEAs exerted a laxative effect via acetylcholine receptors in the mouse constipation model.

  14. Postsynaptic action of brain-derived neurotrophic factor attenuates alpha7 nicotinic acetylcholine receptor-mediated responses in hippocampal interneurons.

    PubMed

    Fernandes, Catarina C; Pinto-Duarte, António; Ribeiro, Joaquim Alexandre; Sebastião, Ana M

    2008-05-21

    Nicotinic mechanisms acting on the hippocampus influence attention, learning, and memory and constitute a significant therapeutic target for many neurodegenerative, neurological, and psychiatric disorders. Here, we report that brain-derived neurotrophic factor (BDNF) (1-100 ng/ml), a member of the neurotrophin gene family, rapidly decreases alpha7 nicotinic acetylcholine receptor responses in interneurons of the hippocampal CA1 stratum radiatum. Such effect is dependent on the activation of the TrkB receptor and involves the actin cytoskeleton; noteworthy, it is compromised when the extracellular levels of the endogenous neuromodulator adenosine are reduced with adenosine deaminase (1 U/ml) or when adenosine A(2A) receptors are blocked with SCH 58261 (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine) (100 nm). The intracellular application of U73122 (1-[6[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) (5 mum), a broad-spectrum inhibitor of phospholipase C, or GF 109203X (bisindolylmaleimide I) (2 mum), a general inhibitor of protein kinase C isoforms, blocks BDNF-induced inhibition of alpha7 nicotinic acetylcholine receptor function. Moreover, in conditions of simultaneous intracellular dialysis of the fast Ca(2+) chelator BAPTA (10 mm) and removal of extracellular Ca(2+) ions, the inhibitory action of BDNF is further prevented. The present findings disclose a novel target for rapid actions of BDNF that might play important roles on synaptic transmission and plasticity in the brain.

  15. Neural Systems Governed by Nicotinic Acetylcholine Receptors: Emerging Hypotheses

    PubMed Central

    Miwa, Julie M.; Freedman, Robert; Lester, Henry A.

    2015-01-01

    Cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) in the brain participate in diverse functions: reward, learning and memory, mood, sensory processing, pain, and neuroprotection. Nicotinic systems also have well-known roles in drug abuse. Here, we review recent insights into nicotinic function, linking exogenous and endogenous manipulations of nAChRs to alterations in synapses, circuits, and behavior. We also discuss how these contemporary advances can motivate attempts to exploit nicotinic systems therapeutically in Parkinson’s disease, cognitive decline, epilepsy, and schizophrenia. PMID:21482353

  16. Synthesis, Trafficking, and Localization of Muscarinic Acetylcholine Receptors

    PubMed Central

    Nathanson, Neil M.

    2008-01-01

    Muscarinic acetylcholine receptors are members of the G-protein coupled receptor superfamily that are expressed in and regulate the function of neurons, cardiac and smooth muscle, glands, and many other cell types and tissues. The correct trafficking of membrane proteins to the cell surface and their subsequent localization at appropriate sites in polarized cells are required for normal cellular signaling and physiological responses. This review will summarize work on the synthesis and trafficking of muscarinic receptors to the plasma membrane and their localization at the cell surface. PMID:18558434

  17. Polyethylene glycol-based homologated ligands for nicotinic acetylcholine receptors☆

    PubMed Central

    Scates, Bradley A.; Lashbrook, Bethany L.; Chastain, Benjamin C.; Tominaga, Kaoru; Elliott, Brandon T.; Theising, Nicholas J.; Baker, Thomas A.; Fitch, Richard W.

    2010-01-01

    A homologous series of polyethylene glycol (PEG) monomethyl ethers were conjugated with three ligand series for nicotinic acetylcholine receptors. Conjugates of acetylaminocholine, the cyclic analog 1-acetyl-4,4-dimethylpiperazinium, and pyridyl ether A-84543 were prepared. Each series was found to retain significant affinity at nicotinic receptors in rat cerebral cortex with tethers of up to six PEG units. Such compounds are hydrophilic ligands which may serve as models for fluorescent/affinity probes and multivalent ligands for nAChR. PMID:19006672

  18. Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197.

    PubMed

    Kim, Jinyeong; Deng, Lili; Hong, Eunsoo; Ryu, Yeonwoo

    2015-12-01

    A novel gene encoding a thermostable esterase (designated as Est-gela) was isolated from the moderate thermophile Bacillus gelatini KACC 12197. The open reading frame of this gene (1170 bp) encodes 389 amino acid residues, and the molecular weight of Est-gela is approximately 42 kDa. The protein sequence of Est-gela shows similarity with β-lactamases and esterases (⩽ 43%). Est-gela contains the Ser-X-X-Lys conserved sequence (Ser58-Met59-Thr60-Lys61) and belongs to family VIII of esterases. We overexpressed Est-gela in Escherichia coli XL1-blue and purified this protein using a His tag. Est-gela showed a strong enzymatic activity toward p-nitrophenyl esters with short acyl chains (⩽ C4) and the strongest activity toward p-nitrophenyl butyrate. Est-gela showed an enhanced enzymatic activity at 65-75 °C and retained more than 90% of the activity after incubation at 65 °C for 180 min. These results indicated that Est-gela was thermostable. In addition, Est-gela showed the maximal activity at pH 10. We also evaluated the effects of surfactants and organic solvents. Surfactants were more effective at improving the enzymatic activity than were organic solvents. Finally, Est-gela hydrolyzed (R,S)-ketoprofen ethyl ester (Kcat/Km = 5.0 ± 0.2 s(-1) mM(-1), mean ± standard error) with enantioselectivity toward (S)-ketoprofen ethyl ester rather than (R)-ketoprofen ethyl ester.

  19. Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei

    PubMed Central

    Yang, Shaoqing; Qin, Zhen; Duan, Xiaojie; Yan, Qiaojuan; Jiang, Zhengqiang

    2015-01-01

    Two hormone-sensitive lipase (HSL) family esterases (RmEstA and RmEstB) from the thermophilic fungus Rhizomucor miehei, exhibiting distinct substrate specificity, have been recently reported to show great potential in industrial applications. In this study, the crystal structures of RmEstA and RmEstB were determined at 2.15 Å and 2.43 Å resolutions, respectively. The structures of RmEstA and RmEstB showed two distinctive domains, a catalytic domain and a cap domain, with the classical α/β-hydrolase fold. Catalytic triads consisting of residues Ser161, Asp262, and His292 in RmEstA, and Ser164, Asp261, and His291 in RmEstB were found in the respective canonical positions. Structural comparison of RmEstA and RmEstB revealed that their distinct substrate specificity might be attributed to their different substrate-binding pockets. The aromatic amino acids Phe222 and Trp92, located in the center of the substrate-binding pocket of RmEstB, blocked this pocket, thus narrowing its catalytic range for substrates (C2–C8). Two mutants (F222A and W92F in RmEstB) showing higher catalytic activity toward long-chain substrates further confirmed the hypothesized interference. This is the first report of HSL family esterase structures from filamentous fungi.jlr The information on structure-function relationships could open important avenues of exploration for further industrial applications of esterases. PMID:26108223

  20. Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters

    PubMed Central

    Martínez-Martínez, Mónica; Lores, Iván; Peña-García, Carlina; Bargiela, Rafael; Reyes-Duarte, Dolores; Guazzaroni, María-Eugenia; Peláez, Ana Isabel; Sánchez, Jesús; Ferrer, Manuel

    2014-01-01

    Herein, we applied a community genomic approach using a naphthalene-enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β-hydrolase family. The protein shares low-to-medium identity (≤ 57%) with known esterase/lipase-like proteins. The enzyme is most active at 25–30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β-hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200–21 000 units g−1 protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio-selectivity (1.0–55 000 units g−1 protein), including (±)-menthyl-acetate, (±)-neomenthyl acetate, (±)-pantolactone, (±)-methyl-mandelate, (±)-methyl-lactate and (±)-glycidyl 4-nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad-spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo-assembly (i.e. through ester bonds) of multi-aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available. PMID:24418210

  1. Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

    PubMed Central

    2014-01-01

    Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274

  2. An integron cassette encoding erythromycin esterase, ere(A), from Providencia stuartii.

    PubMed

    Plante, Isabelle; Centrón, Daniela; Roy, Paul H

    2003-04-01

    We have mapped the variable region of the two class 1 integrons found in the multiresistant strain Providencia stuartii 1723. Integron 1 contains a new arrangement of gene cassettes, aacA4-aadB-aadA1, conferring resistance to all aminoglycosides used for clinical treatment. Integron 2 contains a variant of the gene cassette ere(A), coding for an erythromycin esterase, whose nucleotide sequence shares 93.7% DNA identity with ere(A) from Escherichia coli BM2195 plasmid pIP1100.

  3. A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

    PubMed Central

    Rao, Lang; Xue, Yanfen; Zheng, Yingying; Lu, Jian R.; Ma, Yanhe

    2013-01-01

    Background Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities. Methodology/Principal Findings Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220. Conclusion EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these

  4. A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds.

    PubMed

    Arnling Bååth, Jenny; Giummarella, Nicola; Klaubauf, Sylvia; Lawoko, Martin; Olsson, Lisbeth

    2016-08-01

    The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and (31) P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood.

  5. Substrate-Preference Polymorphism at an Esterase Locus of DROSOPHILA MOJAVENSIS

    PubMed Central

    Zouros, E.; van Delden, W.

    1982-01-01

    In a larval esterase of Drosophila mojavensis there are alleles whose products preferentially hydrolyze α-naphthyl esters, whereas the majority of the alleles hydrolyze preferentially β-naphthyl esters. In a collection of laboratory stocks α alleles have a frequency of 15%. Three different mobilities of α alleles were discovered, suggesting a polymorphism rather than a single mutation event. If substrate-preference polymorphisms are common among "multiple-substrate" enzymes (category II of Gillespie and Langley 1974), allozyme variation at these enzyme loci may well be maintained by balancing selection. PMID:7106559

  6. Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate.

    PubMed

    Fukuda, K; Yamamoto, N; Kiyokawa, Y; Yanagiuchi, T; Wakai, Y; Kitamoto, K; Inoue, Y; Kimura, A

    1998-10-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

  7. Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

    PubMed Central

    Fukuda, Kiyoshi; Yamamoto, Nagi; Kiyokawa, Yoshifumi; Yanagiuchi, Toshiyasu; Wakai, Yoshinori; Kitamoto, Katsuhiko; Inoue, Yoshiharu; Kimura, Akira

    1998-01-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash. PMID:9758847

  8. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

    SciTech Connect

    Wood, S. J.; Li, X.-L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.

    2008-04-01

    The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

  9. Benzoyl-L-arginine methyl ester (BAME)-esterase activity in human plasma during the gravidic-puerperal cycle.

    PubMed

    Salles Meirelles, R

    1977-01-01

    Benzoyl-L-arginine methyl ester (BAME)-esterase activity of plasma was measured in women going through the gravidic-puerperal cycle and compared with plasma of non-pregnant women. Plasma from women in the 36th to 40th week of pregnancy hydrolyzes BAME two times more rapidly than that from non-pregnant women. During pregnancy, BAME-esterase activity in plasma increases progressively up to the 40th week, decreases during labor, and after delivery reaches the same level as in non-pregnant women. The BAME-esterase activity of plasma was affected by the storage temperature, with differences demonstrable between -20 and -4 C and between pregnant and non-pregnant women.

  10. Repetitive nerve stimulation decreases the acetylcholine content of quanta at the frog neuromuscular junction.

    PubMed

    Naves, L A; Van der Kloot, W

    2001-05-01

    We investigated how elevated quantal release produced by motor nerve stimulation affects the size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which excitation-contraction coupling was disrupted. Two hundred stimuli reduced the size of the time integrals of the miniature endplate currents ([integral]MEPCs), measured at the same junction immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When the solution contained 10 microM neostigmine (NEO) 3000 stimuli reduced [integral]MEPCs by 60 %, because with acetylcholinesterase (AChE) inhibited, [integral]MEPC size is more sensitive to changes in acetylcholine (ACh) content. Similar decreases in miniature endplate potential size ([integral]MEPP) followed repetitive stimulation of contracting preparations. The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000 nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less ACh released per quantum. Following 3000 stimuli at 10 Hz the sizes of the [integral]MEPCs increased back to pre-stimulus values with a half-time of 8-10 min. Recovery was blocked by (-)-vesamicol (VES), by hemicholinium-3 (HC3) and by nicotinic cholinergic agonists - all of which inhibit ACh loading into synaptic vesicles. The number of quanta in the total store was estimated by releasing them with carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from unstimulated controls. After resting for hours following stimulation, the releasable number increased, even when ACh loading inhibitors were present. We conclude that the inhibitors do not block a significant fraction of the ACh loading into reformed reserve vesicles and propose that ACh can be loaded in a series of steps.

  11. PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells

    PubMed Central

    Adapala, Ravi K.; Talasila, Phani K.; Bratz, Ian N.; Zhang, David X.; Suzuki, Makoto; Meszaros, J. Gary

    2011-01-01

    Transient receptor potential vanilloid channel 4 (TRPV4) is a polymodally activated nonselective cationic channel implicated in the regulation of vasodilation and hypertension. We and others have recently shown that cyclic stretch and shear stress activate TRPV4-mediated calcium influx in endothelial cells (EC). In addition to the mechanical forces, acetylcholine (ACh) was shown to activate TRPV4-mediated calcium influx in endothelial cells, which is important for nitric oxide-dependent vasodilation. However, the molecular mechanism through which ACh activates TRPV4 is not known. Here, we show that ACh-induced calcium influx and endothelial nitric oxide synthase (eNOS) phosphorylation but not calcium release from intracellular stores is inhibited by a specific TRPV4 antagonist, AB-159908. Importantly, activation of store-operated calcium influx was not altered in the TRPV4 null EC, suggesting that TRPV4-dependent calcium influx is mediated through a receptor-operated pathway. Furthermore, we found that ACh treatment activated protein kinase C (PKC) α, and inhibition of PKCα activity by the specific inhibitor Go-6976, or expression of a kinase-dead mutant of PKCα but not PKCε or downregulation of PKCα expression by chronic 12-O-tetradecanoylphorbol-13-acetate treatment, completely abolished ACh-induced calcium influx. Finally, we found that ACh-induced vasodilation was inhibited by the PKCα inhibitor Go-6976 in small mesenteric arteries from wild-type mice, but not in TRPV4 null mice. Taken together, these findings demonstrate, for the first time, that a specific isoform of PKC, PKCα, mediates agonist-induced receptor-mediated TRPV4 activation in endothelial cells. PMID:21705673

  12. A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone releas...

  13. Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

    PubMed Central

    Rodríguez, María Cecilia; Loaces, Inés; Amarelle, Vanesa; Senatore, Daniella; Iriarte, Andrés; Fabiano, Elena; Noya, Francisco

    2015-01-01

    A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40°C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases. PMID:25973851

  14. Histamine H3 receptors regulate acetylcholine release from the guinea pig ileum myenteric plexus

    SciTech Connect

    Poli, E.; Coruzzi, G.; Bertaccini, G. )

    1991-01-01

    The effect of selective histamine H3-receptor agonists and antagonists on the acetylcholine release from peripheral nerves was evaluated in the guinea pig longitudinal muscle-myenteric plexus preparations, preloaded with ({sup 3}H)choline. In the presence of H1 and H2 blockade, histamine and (R)-{alpha}-methylhistamine inhibited the electrically-evoked acetylcholine release, being (R)-{alpha}-methylhistamine more active than histamine, but behaving as a partial agonist. The effect of histamine was completely reversed by selective H3-blocking drugs, thioperamide and impromidine, while only submaximal doses of (R)-{alpha}-methylhistamine were antagonized. Furthermore, thioperamide and impromidine enhanced the electrically-evoked acetylcholine release. On the contrary, the new H3-blocker, HST-7, was found substantially ineffective, both as histamine antagonist and as acetylcholine overflow enhancer. These data suggest that histamine exerts an inhibitory control on the acetylcholine release from intestinal cholinergic nerves through the activation of H3 receptors.

  15. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    SciTech Connect

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced /sup 155/Eu:/sup 3 +/ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor.

  16. Functional characterization of a novel microbial esterase identified from the Indian Ocean and its use in the stereoselective preparation of ( R)-methyl mandelate

    NASA Astrophysics Data System (ADS)

    Liang, Jiayuan; Sun, Aijun; Zhang, Yun; Deng, Dun; Wang, Yongfei; Ma, Sanmei; Hu, Yunfeng

    2016-11-01

    Genomic mining has identified a novel microbial alkaline esterase from the Indian Ocean. This esterase was overexpressed in E. coli BL21 (DE3) and further functionally characterized. Under optimal conditions (10 mmol/L substrate, pH 6.0, 2 h at 40 °C), this esterase can hydrolyze racemic methyl mandelate to ( R)-methyl mandelate with very high optical purity ( e.e. >99%) and yield (nearly 90%). Interestingly, the stereoselectivity of this esterase is opposite to that of two previously reported lipases that can generate ( S)-methyl mandelate through the hydrolysis of racemic methyl mandelate. No organic solvents or other additives were required to optimize the optical purity and production of the final chiral product ( R)-methyl mandelate, which can potentially simplify the production procedure of ( R)-methyl mandelate catalyzed by esterase.

  17. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

    PubMed Central

    Wood, S. J.; Li, X.-L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.

    2008-01-01

    The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P212121 and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research. PMID:18391420

  18. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina.

    SciTech Connect

    Wood, S. J.; Li, X. -L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.; Biosciences Division; National Center for Agricultural Utilization Research; Slovak Academy of Sciences

    2008-01-01

    The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and X-ray diffraction data were collected to 1.9 {angstrom} resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

  19. The acetylcholine receptor as a cellular receptor for rabies virus.

    PubMed

    Lentz, T L; Burrage, T G; Smith, A L; Tignor, G H

    1983-01-01

    Characterization of specific host cell receptors for enveloped viruses is a difficult problem because many enveloped viruses bind to a variety of substrates which are not obviously related to tissue tropisms in the intact host. Viruses with a limited cellular tropism in infected animals present useful models for studying the mechanisms by which virus attachment regulates the disease process. Rabies virus is a rhabdovirus which exhibits a marked neuronotropism in infected animals. Limited data suggest that spread occurs by transsynaptic transfer of virus. The results of recent experiments at Yale suggest that viral antigen is localized very soon after injection at neuromuscular junctions, the motor nerve endings on muscle tissue. On cultured muscle cells, similar co-localization with the acetylcholine receptor is seen both before and after virus multiplication. Pretreatment of these cells with some ligands of the acetylcholine receptor results in reduced viral infection. These findings suggest that a neurotransmitter receptor or a closely associated molecule may serve as a specific host cell receptor for rabies virus and thus may be responsible for the tissue tropism exhibited by this virus. In addition to clarifying aspects of rabies virus pathogenesis, these studies have broad implications regarding the mechanism by which other viruses or viral immunizations might mediate autoimmune diseases such as myasthenia gravis.

  20. Effects of acetylcholine on neuronal properties in entorhinal cortex

    PubMed Central

    Heys, James G.; Schultheiss, Nathan W.; Shay, Christopher F.; Tsuno, Yusuke; Hasselmo, Michael E.

    2012-01-01

    The entorhinal cortex (EC) receives prominent cholinergic innervation from the medial septum and the vertical limb of the diagonal band of Broca (MSDB). To understand how cholinergic neurotransmission can modulate behavior, research has been directed toward identification of the specific cellular mechanisms in EC that can be modulated through cholinergic activity. This review focuses on intrinsic cellular properties of neurons in EC that may underlie functions such as working memory, spatial processing, and episodic memory. In particular, the study of stellate cells (SCs) in medial entorhinal has resulted in discovery of correlations between physiological properties of these neurons and properties of the unique spatial representation that is demonstrated through unit recordings of neurons in medial entorhinal cortex (mEC) from awake-behaving animals. A separate line of investigation has demonstrated persistent firing behavior among neurons in EC that is enhanced by cholinergic activity and could underlie working memory. There is also evidence that acetylcholine plays a role in modulation of synaptic transmission that could also enhance mnemonic function in EC. Finally, the local circuits of EC demonstrate a variety of interneuron physiology, which is also subject to cholinergic modulation. Together these effects alter the dynamics of EC to underlie the functional role of acetylcholine in memory. PMID:22837741

  1. Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception

    PubMed Central

    Cohen, Emiliano; Chatzigeorgiou, Marios; Husson, Steven J.; Steuer-Costa, Wagner; Gottschalk, Alexander; Schafer, William R.; Treinin, Millet

    2014-01-01

    Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron. PMID:24518198

  2. Corelease of acetylcholine and GABA from cholinergic forebrain neurons

    PubMed Central

    Saunders, Arpiar; Granger, Adam J; Sabatini, Bernardo L

    2015-01-01

    Neurotransmitter corelease is emerging as a common theme of central neuromodulatory systems. Though corelease of glutamate or GABA with acetylcholine has been reported within the cholinergic system, the full extent is unknown. To explore synaptic signaling of cholinergic forebrain neurons, we activated choline acetyltransferase expressing neurons using channelrhodopsin while recording post-synaptic currents (PSCs) in layer 1 interneurons. Surprisingly, we observed PSCs mediated by GABAA receptors in addition to nicotinic acetylcholine receptors. Based on PSC latency and pharmacological sensitivity, our results suggest monosynaptic release of both GABA and ACh. Anatomical analysis showed that forebrain cholinergic neurons express the GABA synthetic enzyme Gad2 and the vesicular GABA transporter (Slc32a1). We confirmed the direct release of GABA by knocking out Slc32a1 from cholinergic neurons. Our results identify GABA as an overlooked fast neurotransmitter utilized throughout the forebrain cholinergic system. GABA/ACh corelease may have major implications for modulation of cortical function by cholinergic neurons. DOI: http://dx.doi.org/10.7554/eLife.06412.001 PMID:25723967

  3. Expression of cloned α6* nicotinic acetylcholine receptors.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Lindstrom, Jon

    2015-09-01

    Nicotinic acetylcholine receptors (AChRs) are ACh-gated ion channels formed from five homologous subunits in subtypes defined by their subunit composition and stoichiometry. Some subtypes readily produce functional AChRs in Xenopus oocytes and transfected cell lines. α6β2β3* AChRs (subtypes formed from these subunits and perhaps others) are not easily expressed. This may be because the types of neurons in which they are expressed (typically dopaminergic neurons) have unique chaperones for assembling α6β2β3* AChRs, especially in the presence of the other AChR subtypes. Because these relatively minor brain AChR subtypes are of major importance in addiction to nicotine, it is important for drug development as well as investigation of their functional properties to be able to efficiently express human α6β2β3* AChRs. We review the issues and progress in expressing α6* AChRs. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

  4. Physiological characterization of human muscle acetylcholine receptors from ALS patients.

    PubMed

    Palma, Eleonora; Inghilleri, Maurizio; Conti, Luca; Deflorio, Cristina; Frasca, Vittorio; Manteca, Alessia; Pichiorri, Floriana; Roseti, Cristina; Torchia, Gregorio; Limatola, Cristina; Grassi, Francesca; Miledi, Ricardo

    2011-12-13

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: "microtransplantation" of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease.

  5. What is the effect of nicotinic acetylcholine receptor stimulation on osteoarthritis in a rodent animal model?

    PubMed Central

    Bock, Kilian; Plaass, Christian; Coger, Vincent; Peck, Claas-Tido; Reimers, Kerstin; Stukenborg-Colsman, Christina; Claassen, Leif

    2016-01-01

    Objectives: Despite the rising number of patients with osteoarthritis, no sufficient chondroprotective and prophylactic therapy for osteoarthritis has been established yet. The purpose of this study was to verify whether stimulation of the nicotinic acetylcholine receptor via nicotine has a beneficial effect on cartilage degeneration in the development of osteoarthritis and is capable of reducing the expression of proinflammatory cytokines and cartilage degrading enzymes in synovial membranes after osteoarthritis induction. Methods: Experimental osteoarthritis was induced in Lewis rats using a standardized osteoarthritis model with monoiodoacetate. A total of 16 Lewis rats were randomized into four groups: control, sham + nicotine application, osteoarthritis, and osteoarthritis + nicotine application. Nicotine (0.625 mg/kg twice daily) was administered intraperitoneally for 42 days. We analyzed histological sections, radiological images and the expression of the proinflammatory cytokines, such as interleukin-1β, tumor necrosis factor-α and interleukin-6, and of matrix metalloproteases 3, 9 and 13 and tissue inhibitors of metalloprotease-1 in synovial membranes via quantitative polymerase chain reaction. Results: Histological and x-ray examination revealed cartilage degeneration in the osteoarthritis group compared to control or sham + nicotine groups (histological control vs osteoarthritis: p = 0.002 and x-ray control vs osteoarthritis: p = 0.004). Nicotine treatment reduced the cartilage degeneration without significant differences. Osteoarthritis induction led to a higher expression of proinflammatory cytokines and matrix metalloproteases as compared to control groups. This effect was attenuated after nicotine administration. The differences of proinflammatory cytokines and matrix metalloproteases did not reach statistical significance. Conclusion: With the present small-scale study, we could not prove a positive effect of nicotinic

  6. Binding of Alpha-Bungarotoxin to Single Identified Neurons of ’Aplysia’ which have Different Ionic Responses to Acetylcholine,

    DTIC Science & Technology

    1976-09-01

    Identifiable Aplysia neurons have one or more of three different ionic responses to acetylcholine, due to Na, Cl, and K conductance increases... Aplysia acetylcholine receptors. Thus the inhibition of the Na response by hexamethonium may be a result of the binding to a site which prevent the conductance change rather than preventing acetylcholine from binding to its receptor.

  7. An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

    PubMed

    Perz, Veronika; Hromic, Altijana; Baumschlager, Armin; Steinkellner, Georg; Pavkov-Keller, Tea; Gruber, Karl; Bleymaier, Klaus; Zitzenbacher, Sabine; Zankel, Armin; Mayrhofer, Claudia; Sinkel, Carsten; Kueper, Ulf; Schlegel, Katharina; Ribitsch, Doris; Guebitz, Georg M

    2016-03-15

    Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/β-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.

  8. Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system

    SciTech Connect

    Hiraki, Toshiki; Shibayama, Naoya; Yoon, Young-Ho; Yun, Kyung-Mook; Hamamoto, Toshiro; Tame, Jeremy R. H.; Park, Sam-Yong

    2007-09-01

    Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2{sub 1}, with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 Å, β = 104.1°. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.3 Å{sup 3} Da{sup −1} and the solvent content is 47%.

  9. Crystal structures of Ophiostoma piceae sterol esterase: structural insights into activation mechanism and product release.

    PubMed

    Gutiérrez-Fernández, Javier; Vaquero, María Eugenia; Prieto, Alicia; Barriuso, Jorge; Martínez, María Jesús; Hermoso, Juan A

    2014-09-01

    Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16-α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30 Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.

  10. Kinetic mechanism of the detoxification of the organophosphate paraoxon by human serum A-esterase.

    PubMed

    Vitarius, J A; Sultatos, L G

    1994-01-01

    The mammalian detoxification of certain organophosphates, such as paraoxon [O,O-diethyl (p-nitrophenyl) phosphate], is catalyzed by the enzyme A-esterase. In this study, incubations of human serum in 50 mM glycine buffer (pH 10.5) with paraoxon resulted in the nonlinear production of p-nitrophenol, characterized by a rapid initial phase for the first several minutes of the incubation, followed by a second, slower phase in which the velocity approached constancy. Production of p-nitrophenol could be accurately fitted to the velocity equation for an Ordered Uni Bi kinetic mechanism with initial-burst activity, yielding estimates of appk2, appk3, and appE, for 10 human subjects. Increasing calcium concentration in the incubation resulted in increases in appk3 and appE, without affecting appk2. Conversely, addition of 1 M sodium chloride decreased the appk3 and appE, but did not alter appk2. And finally, a continuous system computer model was constructed based on the differential equations descriptive of an Ordered Uni Bi kinetic mechanism. This model accurately simulated production of p-nitrophenol from human serum, providing further support that A-esterase hydrolyzes paraoxon by an Ordered Uni Bi kinetic mechanism with initial-burst activity.

  11. Synergistic Enhancement of Cellobiohydrolase Performance on Pretreated Corn Stover by Addition of Xylanase and Esterase Activities

    SciTech Connect

    Selig, M. J.; Knoshaug E. P.; Adney, W. S.; Himmel, M. E.; Decker, S. R.

    2007-11-01

    Significant increases in the depolymerization of corn stover cellulose by cellobiohydrolase I (Cel7A) from Trichoderma reesei were observed using small quantities of non-cellulolytic cell wall-degrading enzymes. Purified endoxylanase (XynA), ferulic acid esterase (FaeA), and acetyl xylan esterase (Axe1) all enhanced Cel7A performance on corn stover subjected to hot water pretreatment. In all cases, the addition of these activities improved the effectiveness of the enzymatic hydrolysis in terms of the quantity of cellulose converted per milligram of total protein. Improvement in cellobiose release by the addition of the non-cellulolytic enzymes ranged from a 13-84% increase over Cel7A alone. The most effective combinations included the addition of both XynA and Axe1, which synergistically enhance xylan conversions resulting in additional synergistic improvements in glucan conversion. Additionally, we note a direct relationship between enzymatic xylan removal in the presence of XynA and the enhancement of cellulose hydrolysis by Cel7A.

  12. Functional Analysis of Esterase TCE2 Gene from Tetranychus cinnabarinus (Boisduval) involved in Acaricide Resistance

    PubMed Central

    Shi, Li; Wei, Peng; Wang, Xiangzun; Shen, Guangmao; Zhang, Jiao; Xiao, Wei; Xu, Zhifeng; Xu, Qiang; He, Lin

    2016-01-01

    The carmine spider mite, Tetranychus cinnabarinus is an important pest of crops and vegetables worldwide, and it has the ability to develop resistance against acaricides rapidly. Our previous study identified an esterase gene (designated TCE2) over-expressed in resistant mites. To investigate this gene’s function in resistance, the expression levels of TCE2 in susceptible, abamectin-, fenpropathrin-, and cyflumetofen-resistant strains were knocked down (65.02%, 63.14%, 57.82%, and 63.99%, respectively) via RNA interference. The bioassay data showed that the resistant levels to three acaricides were significantly decreased after the down-regulation of TCE2, indicating a correlation between the expression of TCE2 and the acaricide-resistance in T. cinnabarinus. TCE2 gene was then re-engineered for heterologous expression in Escherichia coli. The recombinant TCE2 exhibited α-naphthyl acetate activity (483.3 ± 71.8 nmol/mg pro. min−1), and the activity of this enzyme could be inhibited by abamectin, fenpropathrin, and cyflumetofen, respectively. HPLC and GC results showed that 10 μg of the recombinant TCE2 could effectively decompose 21.23% fenpropathrin and 49.70% cyflumetofen within 2 hours. This is the first report of a successful heterologous expression of an esterase gene from mites. This study provides direct evidence that TCE2 is a functional gene involved in acaricide resistance in T. cinnabarinus. PMID:26725309

  13. Diet quality determines lipase gene expression and lipase/esterase activity in Daphnia pulex

    PubMed Central

    Schwarzenberger, Anke; Wacker, Alexander

    2017-01-01

    ABSTRACT We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments. PMID:28069588

  14. Use of esterase activities for the detection of chemical neurotoxic agents.

    PubMed

    Manco, Giuseppe; Nucci, Roberto; Febbraio, Ferdinando

    2009-01-01

    The quest for a quick and easy detection of the neurotoxin levels in the environment has fostered the search for systems alternative to currently employed analytical methods such as spectrophotometer, gas-liquid chromatography, thin-layer chromatography, and more recently mass spectrometry. These drawbacks lead to intense research efforts to develop biosensor devices for the determination of these compounds. In this review, we present an overview of the actual development of research in neurotoxin detection by using enzymatic biosensors based on esterase activity, in particular cholinesterases, and carboxylesterases. Detection by enzymatic activity could be carried out measuring the hydrolysis products or the residual enzymatic activity after inhibition, using a transducer system that makes possible the correlation between the determined activity and the analyte concentration. Several transducer systems were adopted for the neurotoxins identification using esterases, including electrochemical, optical, conductimetric and piezoelectric procedures. The differences in the used transducer determine the final sensitivity and specificity of the biosensor. Moreover, a brief description of immobilization procedure, that is an important step in the biosensor development and could affect the final characteristic of biosensor (sensibility, stability, response time and reproducibility), was accomplished. Final considerations on advantages and problems, related to actual development of these technologies, and its prospective were discussed.

  15. Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

    PubMed

    Asher, O; Lupu-Meiri, M; Jensen, B S; Paperna, T; Fuchs, S; Oron, Y

    1998-07-24

    The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

  16. Paraben esters: review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks.

    PubMed

    Darbre, Philippa D; Harvey, Philip W

    2008-07-01

    This toxicology update reviews research over the past four years since publication in 2004 of the first measurement of intact esters of p-hydroxybenzoic acid (parabens) in human breast cancer tissues, and the suggestion that their presence in the human body might originate from topical application of bodycare cosmetics. The presence of intact paraben esters in human body tissues has now been confirmed by independent measurements in human urine, and the ability of parabens to penetrate human skin intact without breakdown by esterases and to be absorbed systemically has been demonstrated through studies not only in vitro but also in vivo using healthy human subjects. Using a wide variety of assay systems in vitro and in vivo, the oestrogen agonist properties of parabens together with their common metabolite (p-hydroxybenzoic acid) have been extensively documented, and, in addition, the parabens have now also been shown to possess androgen antagonist activity, to act as inhibitors of sulfotransferase enzymes and to possess genotoxic activity. With the continued use of parabens in the majority of bodycare cosmetics, there is a need to carry out detailed evaluation of the potential for parabens, together with other oestrogenic and genotoxic co-formulants of bodycare cosmetics, to increase female breast cancer incidence, to interfere with male reproductive functions and to influence development of malignant melanoma which has also recently been shown to be influenced by oestrogenic stimulation.

  17. Methamphetamine exposure during brain development alters the brain acetylcholine system in adolescent mice.

    PubMed

    Siegel, Jessica A; Park, Byung S; Raber, Jacob

    2011-10-01

    Children exposed to methamphetamine during brain development as a result of maternal drug use have long-term hippocampus-dependent cognitive impairments, but the mechanisms underlying these impairments are not understood. The acetylcholine system plays an important role in cognitive function and potential methamphetamine-induced acetylcholine alterations may be related to methamphetamine-induced cognitive impairments. In this study, we investigated the potential long-term effects of methamphetamine exposure during hippocampal development on the acetylcholine system in adolescence mice on postnatal day 30 and in adult mice on postnatal day 90. Methamphetamine exposure increased the density of acetylcholine neurons in regions of the basal forebrain and the area occupied by acetylcholine axons in the hippocampus in adolescent female mice. In contrast, methamphetamine exposure did not affect the density of GABA cells or total neurons in the basal forebrain. Methamphetamine exposure also increased the number of muscarinic acetylcholine receptors in the hippocampus of adolescent male and female mice. Our results demonstrate for the first time that methamphetamine exposure during hippocampal development affects the acetylcholine system in adolescent mice and that these changes are more profound in females than males.

  18. Acetylcholine receptor extracellular domain determines sensitivity to nicotine-induced inactivation.

    PubMed

    Kuryatov, A; Olale, F A; Choi, C; Lindstrom, J

    2000-03-30

    We have shown previously that chronic exposure to submicromolar concentrations of nicotine permanently inactivates alpha4beta2 and alpha7 neuronal nicotinic acetylcholine receptors while alpha3beta2 acetylcholine receptors are resistant to inactivation. Phosphorylation of the large cytoplasmic domain has been proposed to mediate functional inactivation. Chimeric subunits consisting of human alpha4 sequence from their N-terminus to either the beginning of the first transmembrane domain or the large cytoplasmic domain and alpha3 sequences thereafter formed acetylcholine receptors with beta2 subunits which were as susceptible to nicotine-induced inactivation as wild-type alpha4 acetylcholine receptors. The converse chimeras, containing the N-terminal parts of the alpha3 subunit and the C-terminal parts of the alpha4 subunit, formed acetylcholine receptors with beta2 subunits which were as resistant to nicotine-induced inactivation as wild-type alpha3beta2 acetylcholine receptors. Thus, inactivation of acetylcholine receptors produced by chronic exposure to nicotine results primarily from effects of the agonist on the extracellular and transmembrane domains of the alpha subunit.

  19. Specific Stimulated Uptake of Acetylcholine by Torpedo Electric Organ Synaptic Vesicles

    NASA Astrophysics Data System (ADS)

    Parsons, Stanley M.; Koenigsberger, Robert

    1980-10-01

    The specificity of acetylcholine uptake by synaptic vesicles isolated from the electric organ of Torpedo californica was studied. In the absence of cofactors, [3H]acetylcholine was taken up identically to [14C]choline in the same solution (passive uptake), and the equilibrium concentration achieved inside the vesicles was equal to the concentration outside. In the presence of MgATP, [3H]acetylcholine and [14C]choline in the same solution were taken up identically, except only about half as much of each was taken up (suppressed uptake). [3H]Acetylcholine uptake was stimulated by MgATP and HCO3 about 4-fold relative to suppressed uptake, for a net concentrative uptake of about 2:1 (stimulated uptake). Uptake of [14C]choline in the same solution remained at the suppressed level. [3H]Acetylcholine taken up under stimulated conditions migrated with vesicles containing [14C]mannitol on analytical glycerol density gradients during centrifugation. Vesicles were treated with nine protein modification reagents under mild conditions. Two reagents had no effect on, dithiothreitol potentiated, and six reagents strongly inhibited subsequent stimulated uptake of [3H]acetylcholine. The results indicate that uptake of acetylcholine is conditionally specific for the transported substrate, is carried out by the synaptic vesicles rather than a contaminant of the preparation, and requires a functional protein system containing a critical sulfhydryl group.

  20. Novel ferulate esterase from Gram-positive lactic acid bacteria and analyses of the recombinant enzyme produced in E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a plate containing ethyl ferulate as sole carbon source, various bacteria cultures were screened for ferulate esterase (FAE). Among a dozen of species showing positive FAE, one Lactobacillus fermentum strain NRRL 1932 demonstrated the strongest activity. Using a published sequence of ferulate ...

  1. Novel feruloyl esterase from Lactobacillus fermentum NRRL B-1932 and analysis of the recombinant enzyme produced in Escherichia coli.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using agar plates containing ethyl ferulate as the sole carbon source, 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity. Among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate, Lactobacillus fermentum NRRL B-1932 demonstrated the stronge...

  2. Monitoring Lipase/Esterase Activity by Stopped Flow in a Sequential Injection Analysis System Using p-Nitrophenyl Butyrate

    PubMed Central

    Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J.

    2015-01-01

    Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05–1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed. PMID:25633600

  3. Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate.

    PubMed

    Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J

    2015-01-27

    Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05-1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed.

  4. Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.

    PubMed

    Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J; Madrid, Susan M; Brinch-Pedersen, Henrik; Holm, Preben B; Scheller, Henrik V

    2010-04-01

    Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

  5. Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

    SciTech Connect

    Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J.; Madrid, Susan M.; Brinch-Pedersen, Henrik; Holm, Preben B.; Scheller, Henrik V.

    2009-12-08

    Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and three fold relative to wild type. The grains were shriveled and had a 25-33% decrease in mass. Extensive analysis of the cell walls showed a 10-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water extractable arabinoxylan, and a shift in the MW of the water extractable arabinoxylan from being mainly larger than 85 kD to being between 2 kD and 85 kD. Ferulic acid esterase expressing grains were also shriveled and the seed weight was decreased by 20-50%. No ferulic acid esterase activity could be detected in wild type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15-40% increase in water unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13 and 34%. In all the plants the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

  6. Correlation between esterase electrophoretic polymorphism and virulence-associated traits in extra-intestinal invasive strains of Escherichia coli.

    PubMed Central

    Goullet, P.; Picard, B.; Contrepois, M.; De Rycke, J.; Barnouin, J.

    1994-01-01

    The electrophoretic variations of carboxylesterase B and of esterases A, C and I, the presence of mannose resistant haemagglutinin, alpha-haemolysin, cytotoxic necrotizing factor type 1 (CNF1) and certain O antigens were compared in 150 strains of Escherichia coli responsible for extra-intestinal infections. Electrophoretic mobilities of outer membrane proteins (OMP) were also studied for strains belonging to O4, O6, O7, O8 and O75 serogroups. Fast migrating allozymes of carboxylesterase B (pattern B1) were correlated with slow migrating allozymes of esterase C, serogroups O7 and O8, lack of virulence factor, and particular OMP patterns, whereas slow migrating allozymes of carboxylesterase B (pattern B2) were correlated with fast migrating allozymes of esterase C, serogroups O2, O4, O6, O18 and O75, virulence factor production, and distinct OMP patterns. Allozymes of esterases A and I were not clearly correlated with the distribution of virulence factors. The pattern B2 was more strongly associated with CNF1 than with alpha-haemolysin and mannose resistant haemagglutinin. These results substantiate the view that the electrophoretic pattern B2 of carboxylesterase B identified most of the highly pathogenic strains implicated in extra-intestinal infection of humans. Images Fig. 2 PMID:7509755

  7. Crystallization and Preliminary X-ray Diffraction Analysis of the Glucuronoyl Esterase Catalytic Domain from Hypocrea jecorina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was over-expressed, purified, and crystallized by sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. Crystals had space group P212121 and X-ray diffraction data were...

  8. Identification and functional characterization of esterases in Euschistus heros (Hemiptera, Pentatomidae) and their relationship with thiamethoxam and lambda-cyhalothrin.

    PubMed

    Hegeto, L A; Ronqui, L; Lapenta, A S; Albuquerque, F A

    2015-09-22

    The brown stink bug Euschistus heros is the most abundant species of the soybean-sucking bugs, and causes large economic losses. Applying different chemical groups of organosynthetic insecticides for its control increases the potential for resistance. Esterases are a group of enzymes that play a variety of roles in insects, and some of them are related to the metabolism of xenobiotics. The aim of this study was to analyze the esterase isoenzyme system of this species and investigate its response to Engeo™ Pleno (thiamethoxam and lambda-cyhalothrin), which is the most widely used pesticide in soybean crops. Two strains were analyzed: the EB strain, which had been free of insecticides for several generations; and the MA strain, which was collected in a location exposed to agrochemicals. By analyzing the polyacrylamide gel electrophoresis profile, seven different esterases in adults and nymphs of both strains were found. Eight gene loci were responsible for the synthesis of these enzymes. The differences in esterases between the two strains and enzyme changes in insects exposed to Engeo™ Pleno suggest that EST-2 and EST-4 are related to the metabolism of the agrochemical used and are mechanisms of resistance.

  9. Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 ...

  10. ISOLATION OF JUVENILE HORMONES ESTERASE AND ITS PARTIAL CDNA CLONE FROM THE BEETLE, TENEBRIO MOLITOR. (R825433)

    EPA Science Inventory

    Juvenile hormone esterase (JHE) plays an essential role in insect development. It is partially responsible for the clearance of juvenile hormone (JH) which regulates various aspects of insect development and reproduction. Because of its role in regulating JH titer, this enzyme...

  11. Structure and properties of the esterase from non-LTR retrotransposons suggest a role for lipids in retrotransposition

    PubMed Central

    Schneider, Anna M.; Schmidt, Steffen; Jonas, Stefanie; Vollmer, Benjamin; Khazina, Elena; Weichenrieder, Oliver

    2013-01-01

    Non-LTR retrotransposons are mobile genetic elements and play a major role in eukaryotic genome evolution and disease. Similar to retroviruses they encode a reverse transcriptase, but their genomic integration mechanism is fundamentally different, and they lack homologs of the retroviral nucleocapsid-forming protein Gag. Instead, their first open reading frames encode distinct multi-domain proteins (ORF1ps) presumed to package the retrotransposon-encoded RNA into ribonucleoprotein particles (RNPs). The mechanistic roles of ORF1ps are poorly understood, particularly of ORF1ps that appear to harbor an enzymatic function in the form of an SGNH-type lipolytic acetylesterase. We determined the crystal structures of the coiled coil and esterase domains of the ORF1p from the Danio rerio ZfL2-1 element. We demonstrate a dimerization of the coiled coil and a hydrolytic activity of the esterase. Furthermore, the esterase binds negatively charged phospholipids and liposomes, but not oligo-(A) RNA. Unexpectedly, the esterase can split into two dynamic half-domains, suited to engulf long fatty acid substrates extending from the active site. These properties indicate a role for lipids and membranes in non-LTR retrotransposition. We speculate that Gag-like membrane targeting properties of ORF1ps could play a role in RNP assembly and in membrane-dependent transport or localization processes. PMID:24003030

  12. Stimulation of the Nonneuronal Cholinergic System by Highly Diluted Acetylcholine in Keratinocytes.

    PubMed

    Uberti, Francesca; Bardelli, Claudio; Morsanuto, Vera; Ghirlanda, Sabrina; Cochis, Andrea; Molinari, Claudio

    2017-01-01

    The physiological effects of acetylcholine on keratinocytes depend on the presence of nicotinic and muscarinic receptors. The role of nonneuronal acetylcholine in keratinocytes could have important clinical implications for patients with various skin disorders such as nonhealing wounds. In order to evaluate the efficacy of highly diluted acetylcholine solutions obtained by sequential kinetic activation, we aimed to investigate the effects of these solutions on normal human keratinocytes. Two different concentrations (10 fg/mL and 1 pg/mL) and formulations (kinetically activated and nonkinetically activated) of acetylcholine were used to verify keratinocyte viability, proliferation, and migration and the intracellular pathways involved using MTT, crystal violet, wound healing, and Western blot compared to 147 ng/mL acetylcholine. The activated formulations (1 pg/mL and 10 fg/mL) revealed a significant capacity to increase migration, cell viability, and cell proliferation compared to 147 ng/mL acetylcholine, and these effects were more evident after a single administration. Sequential kinetic activation resulted in a statistically significant decrease in reactive oxygen species production accompanied by an increase in mitochondrial membrane potential and a decrease in oxygen consumption compared to 147 ng/mL acetylcholine. The M1 muscarinic receptor was involved in these effects. Finally, the involvement of ERK/mitogen-activated protein kinases (MAPK) and KI67 confirmed the effectiveness of the single treatment on cell proliferation. The intracellular pathways of calcium were investigated as well. Our results indicate for the first time that highly diluted and kinetically activated acetylcholine seems to play an active role in an in vitro model of wound healing. Moreover, the administration of acetylcholine within the physiological range may not only be effective but is also likely to be safe.

  13. An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase (Xy1D) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls.

    PubMed Central

    Bartolomé, B; Faulds, C B; Kroon, P A; Waldron, K; Gilbert, H J; Hazlewood, G; Williamson, G

    1997-01-01

    Diferulate esters strengthen and cross-link primary plant cell walls and help to defend the plant from invading microbes. Phenolics also limit the degradation of plant cell walls by saprophytic microbes and by anaerobic microorganisms in the rumen. We show that incubation of wheat and barley cell walls with ferulic acid esterase from Aspergillus niger (FAE-III) or Pseudomonas fluorescens (Xy1D), together with either xylanase I from Aspergillus niger, Trichoderma viride xylanase, or xylanase from Pseudomonas fluorescens (XylA), leads to release of the ferulate dimer 5-5' diFA [(E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid]. Direct saponification of the cell walls without enzyme treatment released the following five identifiable ferulate dimers (in order of abundance): (Z)-beta-(4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy)-4-hydroxy-3-methoxycinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl) -7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid, 5-5' diFA, (E,E)-4, 4'-dihydroxy-3, 5'-dimethoxy-beta, 3'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -6-methoxy-1, 2-dihydronaphthalene-2, 3-dicarboxylic acid. Incubation of the wheat or barley cell walls with xylanase, followed by saponification of the solubilized fraction, yielded 5-5'diFA and, in some cases, certain of the above dimers, depending on the xylanase used. These experiments demonstrate that FAE-III and XYLD specifically release only esters of 5-5'diFA from either xylanase-treated or insoluble fractions of cell walls, even though other esterified dimers were solubilized by preincubation with xylanase. It is also concluded that the esterified dimer content of the xylanase-solubilized fraction depends on the source of the xylanase. PMID:8979352

  14. Angiogenesis Inhibitors

    MedlinePlus

    ... inhibitors: current strategies and future prospects. CA: A Cancer Journal for Clinicians 2010; 60(4):222–243. [PubMed Abstract] Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nature Reviews Clinical Oncology 2009; 6(8):465– ...

  15. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  16. A model of the human M2 muscarinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Jöhren, Kirstin; Höltje, Hans-Dieter

    2002-11-01

    The M2 muscarinic acetylcholine receptor belongs to the family of rhodopsin like G-Protein Coupled Receptors. This subtype of muscarinic receptors is of special interest because it bears, aside from an orthosteric binding site, also an allosteric binding site. Based on the X-ray structure of bovine rhodopsin a complete homology model of the human M2 receptor was developed. For the orthosteric binding site point mutations and binding studies with different agonists and antagonists are available. This knowledge was utilized for an initial verification of the M2 model. Allosteric modulation of activity is mediated by structurally different ligands such as gallamine, caracurine V salts or W84 (a hexamethonium-derivative). Caracurine V derivatives with different affinities to M2 were docked using GRID-fields. Subsequent molecular dynamics simulations yielded different binding energies based on diverse electrostatic and lipophilic interactions. The calculated affinities are in good agreement to experimentally determined affinities.

  17. Caffeine potentiates the enhancement by choline of striatal acetylcholine release

    NASA Technical Reports Server (NTRS)

    Johnson, D. A.; Ulus, I. H.; Wurtman, R. J.

    1992-01-01

    We investigated the effect of peripherally administered caffeine (50 mg/kg), choline (30, 60, or 120 mg/kg) or combinations of both drugs on the spontaneous release of acetylcholine (ACh) from the corpus striatum of anesthetized rats using in vivo microdialysis. Caffeine alone or choline in the 30 or 60 mg/kg dose failed to increase ACh in microdialysis samples; the 120 mg/kg choline dose significantly enhanced ACh during the 80 min following drug administration. Coadministration of caffeine with choline significantly increased ACh release after each of the choline doses tested. Peak microdialysate levels with the 120 mg/kg dose were increased 112% when caffeine was additionally administered, as compared with 54% without caffeine. These results indicate that choline administration can enhance spontaneous ACh release from neurons, and that caffeine, a drug known to block adenosine receptors on these neurons, can amplify the choline effect.

  18. Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors.

    PubMed

    Kasheverov, Igor E; Utkin, Yuri N; Tsetlin, Victor I

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake alpha-neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to alpha-neurotoxins, and polypeptide neurotoxins waglerins from the venom of Wagler's pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimer's disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine-binding protein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.

  19. Characterization and photoaffinity labeling of the muscarinic acetylcholine receptor

    SciTech Connect

    Cremo, C.R.

    1983-01-01

    The muscarinic acetylcholine receptor, identified by tritiated L-quinuclidinyl benzilate (L-(/sup 3/H)QNB) binding, was solubilized from porcine atrial membranes using a 5:1 (w/w) ratio of digitonin and cholate. Specific binding activities of the solubilized receptor solutions usually exceeded 1.0 nmol L-(/sup 3/H)QNB sites per gram of protein, representing 75-98% total site recovery and a two- to three-fold enrichment over untreated atrial membranes. Two rapid assays for measuring the binding activities of detergent extracts were devised and compared with equilibrium dialysis. All three methods gave similar results. The equilibrium dissociation constant of the solubilized receptor for L-(/sup 3/H)QNB as determined by the three methods varied from 230 to 450 pM depending on the method and temperature. The interaction of alkyl quanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkyl guanidines with alkyl chain lengths from one to ten carbons displaced (/sup 3/H)L-quinuclidinyl bensilate ((/sup 3/H)L-QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of -1n K/sub i/ versus alkyl carbon chain number, a value of -(473 +/- 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The synthesis and properties of a radiolabeled muscarinic antagonist photoaffinity probe, (/sup 3/H) p-azidoatropine methyl iodide were reported.

  20. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    SciTech Connect

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K.

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  1. Morphine Increases Acetylcholine Release in the Trigeminal Nuclear Complex

    PubMed Central

    Zhu, Zhenghong; Bowman, Heather R.; Baghdoyan, Helen A.; Lydic, Ralph

    2008-01-01

    Study Objectives: The trigeminal nuclear complex (V) contains cholinergic neurons and includes the principal sensory trigeminal nucleus (PSTN) which receives sensory input from the face and jaw, and the trigeminal motor nucleus (MoV) which innervates the muscles of mastication. Pain associated with pathologies of V is often managed with opioids but no studies have characterized the effect of opioids on acetylcholine (ACh) release in PSTN and MoV. Opioids can increase or decrease ACh release in brainstem nuclei. Therefore, the present experiments tested the 2-tailed hypothesis that microdialysis delivery of opioids to the PSTN and MoV significantly alters ACh release. Design: Using a within-subjects design and isoflurane-anesthetized Wistar rats (n = 53), ACh release in PSTN during microdialysis with Ringer's solution (control) was compared to ACh release during dialysis delivery of the sodium channel blocker tetrodotoxin, muscarinic agonist bethanechol, opioid agonist morphine, mu opioid agonist DAMGO, antagonists for mu (naloxone) and kappa (nor-binaltorphimine; nor-BNI) opioid receptors, and GABAA antagonist bicuculline. Measurements and Results: Tetrodotoxin decreased ACh, confirming action potential-dependent ACh release. Bethanechol and morphine caused a concentration-dependent increase in PSTN ACh release. The morphine-induced increase in ACh release was blocked by nor-BNI but not by naloxone. Bicuculline delivered to the PSTN also increased ACh release. ACh release in the MoV was increased by morphine, and this increase was not blocked by naloxone or nor-BNI. Conclusions: These data comprise the first direct measures of ACh release in PSTN and MoV and suggest synaptic disinhibition as one possible mechanism by which morphine increases ACh release in the trigeminal nuclei. Citation: Zhu Z; Bowman HR; Baghdoyan HA; Lydic R. Morphine increases acetylcholine release in the trigeminal nuclear complex. SLEEP 2008;31(12):1629–1637. PMID:19090318

  2. Physiological characterization of human muscle acetylcholine receptors from ALS patients

    PubMed Central

    Palma, Eleonora; Inghilleri, Maurizio; Conti, Luca; Deflorio, Cristina; Frasca, Vittorio; Manteca, Alessia; Pichiorri, Floriana; Roseti, Cristina; Torchia, Gregorio; Limatola, Cristina; Grassi, Francesca; Miledi, Ricardo

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: “microtransplantation” of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease. PMID:22128328

  3. Effects of nonylphenol on the calcium signal and catecholamine secretion coupled with nicotinic acetylcholine receptors in bovine adrenal chromaffin cells.

    PubMed

    Liu, Pei-Shan; Liu, Ging-Hui; Chao, Wei-Liang

    2008-02-03

    Nonylphenol (NP) is the most critical metabolite of alkylphenol polyethoxylate detergents. NP is known as an endocrine disruptor with estrogenic activities and as an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Estrogen has modulatory roles on ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs). Ca(2+)-ATPase inhibitors can modulate the cytosolic calcium concentration ([Ca(2+)](c)]) and thus can affect the calcium signaling coupled with nAChRs. Therefore, NP is predicted to have complex effects on the Ca(2+) signaling and secretion coupled with nAChRs. This study investigated these effects using bovine adrenal chromaffin cells. The results show that NP suppressed the Ca(2+) signaling coupled with nAChRs and voltage-operated Ca(2+) channels in a dose-dependent manner, with IC(50)s of 1 and 5.9 microM, respectively. Estradiol exhibits similar suppression but much lower inhibitory potencies. NP alone induced a transient rise in [Ca(2+)](c) in the presence or absence of extracellular calcium. Thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, partially suppressed the [Ca(2+)](c) rise induced by NP, but NP totally blocked the [Ca(2+)](c) rise induced by thapsigargin. This illustrates that NP can cause Ca(2+) release from thapsigargin-insensitive pools. Thapsigargin suppressed the Ca(2+) signaling coupled with nAChRs but increased that coupled with voltage-operated Ca(2+) channels. We propose that three routes are responsible for the effects of NP on nAChRs: named receptor channels, voltage-gated Ca(2+) channels, and Ca(2+)-induced Ca(2+) release. Three routes are related to the characteristics of NP as steroid-like compounds and Ca(2+)-ATPase inhibitor.

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

    PubMed

    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

  5. Acetylcholine receptors at neuromuscular synapses: phylogenetic differences detected by snake alpha-neurotoxins.

    PubMed Central

    Burden, S J; Hartzell, H C; Yoshikami, D

    1975-01-01

    Phylogenetic differences in acetylcholine receptors from skeletal neuromuscular synapses of various species of snakes and lizards have been investigated, using the snake venom alpha-neurotoxins alpha-atratoxin (cobrotoxin) and alpha-bungarotoxin. The acetylcholine receptors of the phylogenetically primitive lizards, like those from all other vertebrates previously tested, are blocked by these alpha-neurotoxins. In contrast, receptors from snakes and advanced lizards are insensitive to one or both of the toxins. It is suggested that toxin-resistant acetylcholine receptors appeared early in the evolution of Squamata and preceded the appearance of alpha-neurotoxins. Images PMID:1081230

  6. Immunological relationship between acetylcholine receptor and thymus: a possible significance in myasthenia gravis.

    PubMed Central

    Aharonov, A; Tarrab-Hazdai, R; Abramsky, O; Fuchs, S

    1975-01-01

    A defined immunological cross-reaction was observed between acetylcholine receptor fraction from the electric eel, Electrophorus electricus, and two calf thymus fractions. The cross-reaction was demonstrated on the cellular level by means of the lymphocyte transformation technique, and on the humoral level, by means of the microcomplement fixation assay. In the human disease myasthenia gravis both acetylcholine receptor at the neuromuscular junction and the thymus are affected, probably by an autoimmune mechanism. The immunological cross-reaction between acetylcholine receptor and thymic components may explain the association between endplate and thymus disorders in myasthenia gravis. PMID:1055418

  7. Base-catalyzed and cholinesterase-catalyzed hydrolysis of acetylcholine and optically active analogs.

    PubMed

    Schowen, K B; Smissman, E E; Stephen, W F

    1975-03-01

    The base- and cholinestrase-catalyzed hydrolyses of the following optically active analogs of acetylcholine were studied: 3 (a)-trimethylammonium-2(a)-acetoxy-trans-decalin iodide, threo- and erythro-alpha, beta-dimethylacetylcholine iodide, alpha-methylacetylcholine, and beta-methylacetylcholine. Evidence that the optimum dihedral +N-C-C-O angle in the transition state for acetylcholinesterase hydrolysis of acetylcholine analogs is positive and anticlinal is given. The data obtained suggest that acetylcholine undergoes a geometrically flexible mode of attachment to the enzyme.

  8. Synergistic effect of choline and carnitine on acetylcholine synthesis in neuroblastoma NB-2a cells.

    PubMed

    Wawrzeńczyk, A; Nałecz, K A; Nałecz, M J

    1994-07-15

    An influence of carnitine on acetylcholine synthesis from radiolabeled glucose was monitored in neuroblastoma NB-2a cells. Upon addition of carnitine the distribution of its derivatives was found significantly different than the values published for brain, the level of long-chain acyl derivatives being much higher and reaching 60%. Carnitine itself did not change acetylcholine level. Together with choline (20 microM), carnitine was observed to stimulate (by 36%) acetylcholine synthesis in a synergistic way, which indicated that both substrates could be limiting factors of this process in NB-2a cell line of neuroblastoma.

  9. A cold-adapted esterase of a novel marine isolate, Pseudoalteromonas arctica: gene cloning, enzyme purification and characterization.

    PubMed

    Al Khudary, Rami; Venkatachalam, Ramprasath; Katzer, Moritz; Elleuche, Skander; Antranikian, Garabed

    2010-05-01

    A gene encoding an esterase (estO) was identified and sequenced from a gene library screen of the psychrotolerant bacterium Pseudoalteromonas arctica. Analysis of the 1,203 bp coding region revealed that the deduced peptide sequence is composed of 400 amino acids with a predicted molecular mass of 44.1 kDa. EstO contains a N-terminal esterase domain and an additional OsmC domain at the C-terminus (osmotically induced family of proteins). The highly conserved five-residue motif typical for all alpha/beta hydrolases (G x S x G) was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser(106), Asp(196), and His(225). Sequence comparison showed that EstO exhibits 90% amino acid identity with hypothetical proteins containing similar esterase and OsmC domains but only around 10% identity to the amino acid sequences of known esterases. EstO variants with and without the OsmC domain were produced and purified as His-tag fusion proteins in E. coli. EstO displayed an optimum pH of 7.5 and optimum temperature of 25 degrees C with more than 50% retained activity at the freezing point of water. The thermostability of EstO (50% activity after 5 h at 40 degrees C) dramatically increased in the truncated variant (50% activity after 2.5 h at 90 degrees C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain fatty acids (C(2)-C(8)).

  10. Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage

    PubMed Central

    Santo-Orihuela, Pablo Luis; Carvajal, Guillermo; Picollo, María Inés; Vassena, Claudia Viviana

    2013-01-01

    The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia) and from domiciliary areas, including El Palmar (Bolivia) and La Pista (Argentina). Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL) were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR) value (44.90) compared to the susceptible reference strain (NFS), whereas the Veinte de Octubre samples had the lowest value (0.50). Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP) on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein) and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively). The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP). PMID:24402155

  11. Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regulates EGR-1 and REST expression in neuroblastoma cells.

    PubMed

    Salani, Monica; Anelli, Tonino; Tocco, Gabriella Augusti; Lucarini, Elena; Mozzetta, Chiara; Poiana, Giancarlo; Tata, Ada Maria; Biagioni, Stefano

    2009-02-01

    Neurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase-transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1-M4 subtypes and the activation of both IP(3) and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor-1 (EGR-1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR-1 induction. The role of EGR-1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR-1. EGR-1 clones show increased neurite extension and a decrease in Repressor Element-1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc-finger domain, a dominant negative inhibitor of EGR-1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR-1 and REST transcription factors.

  12. Syntheses and Radiosyntheses of Two Carbon-11 Labeled Potent and Selective Radioligands for Imaging Vesicular Acetylcholine Transporter

    PubMed Central

    Padakanti, Prashanth K.; Zhang, Xiang; Li, Junfeng; Parsons, Stanley M.; Perlmutter, Joel S.; Tu, Zhude

    2015-01-01

    Purpose The vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. The syntheses and C-11 labeling of two potent enantiopure VAChT inhibitors are reported here. Procedures Two VAChT inhibitors, (±)-2 and (±)-6, were successfully synthesized. A chiral HPLC column was used to resolve the enantiomers from each corresponding racemic mixture for in vitro characterization. The radiosyntheses of (−)-[11C]2 and (−)-[11C]6 from the corresponding desmethyl phenol precursor was accomplished using [11C]methyl iodide or [11C]methyl triflate, respectively. Results The synthesis of (−)-[11C]2 was accomplished with 40–50 % radiochemical yield (decay-corrected), SA>480 GBq/μmol (EOB), and radiochemical purity >99 %. Synthesis of (−)-[11C]6 was accomplished with 5–10 % yield, SA>140 GBq/μmol (EOB), and radiochemical purity >97 %. The radiosynthesis and dose formulation of each tracer was completed in 55–60 min. Conclusions Two potent enantiopure VAChT ligands were synthesized and 11C-labeled with good radiochemical yield and specific activity. PMID:24875230

  13. Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

    PubMed

    Haruki, M; Oohashi, Y; Mizuguchi, S; Matsuo, Y; Morikawa, M; Kanaya, S

    1999-07-09

    Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.

  14. Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli.

    PubMed

    Ounissi, H; Courvalin, P

    1985-01-01

    We have cloned and determined the nucleotide sequence of the gene ereA of plasmid pIP1100 which confers high-level resistance to erythromycin (Em) in Escherichia coli. The gene was defined by initiation and termination codons and by in vitro insertion-inactivation into an open reading frame (ORF) of 1032 bp corresponding to a product with an Mr of 37 765. However, the enzyme, an Em esterase, displayed an apparent Mr of 43 000 upon electrophoresis of a minicell extract on the SDS-polyacrylamide gels. The G + C content (50.5%) of the gene ereA and the preferential codon usage in its ORF suggest that this resistance determinant should be indigenous to E. coli.

  15. Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus.

    PubMed

    Damásio, André R L; Braga, Cleiton Márcio Pinto; Brenelli, Lívia B; Citadini, Ana Paula; Mandelli, Fernanda; Cota, Junio; de Almeida, Rodrigo Ferreira; Salvador, Victor Hugo; Paixao, Douglas Antonio Alvaredo; Segato, Fernando; Mercadante, Adriana Zerlotti; de Oliveira Neto, Mario; do Santos, Wanderley Dantas; Squina, Fabio M

    2013-08-01

    The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.

  16. Feruloyl esterases from Schizophyllum commune to treat food industry side-streams.

    PubMed

    Nieter, Annabel; Kelle, Sebastian; Linke, Diana; Berger, Ralf G

    2016-11-01

    Agro-industrial side-streams are abundant and renewable resources of hydroxycinnamic acids with potential applications as antioxidants and preservatives in the food, health, cosmetic, and pharmaceutical industries. Feruloyl esterases (FAEs) from Schizophyllum commune were functionally expressed in Pichia pastoris with extracellular activities of 6000UL(-1). The recombinant enzymes, ScFaeD1 and ScFaeD2, released ferulic acid from destarched wheat bran and sugar beet pectin. Overnight incubation of coffee pulp released caffeic (>60%), ferulic (>80%) and p-coumaric acid (100%) indicating applicability for the valorization of food processing wastes and enhanced biomass degradation. Based on substrate specificity profiling and the release of diferulates from destarched wheat bran, the recombinant FAEs were characterized as type D FAEs. ScFaeD1 and ScFaeD2 preferably hydrolyzed feruloylated saccharides with ferulic acid esterified to the O-5 position of arabinose residues and showed an unprecedented ability to hydrolyze benzoic acid esters.

  17. Rapid method for identification of macrophages in suspension by acid alpha-naphthyl acetate esterase activity.

    PubMed

    Ennist, D L; Jones, K H

    1983-07-01

    A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.

  18. Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

    SciTech Connect

    Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

    1994-12-31

    The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which is known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.

  19. Structure of EstA esterase from psychrotrophic Pseudoalteromonas sp. 643A covalently inhibited by monoethylphosphonate

    SciTech Connect

    Brzuszkiewicz, Anna; Nowak, Elzbieta; Dauter, Zbigniew; Dauter, Miroslawa; Cieslinski, Hubert; Dlugolecka, Anna; Kur, Józef

    2010-10-28

    The crystal structure of the esterase EstA from the cold-adapted bacterium Pseudoalteromonas sp. 643A was determined in a covalently inhibited form at a resolution of 1.35 {angstrom}. The enzyme has a typical SGNH hydrolase structure consisting of a single domain containing a five-stranded {beta}-sheet, with three helices at the convex side and two helices at the concave side of the sheet, and is ornamented with a couple of very short helices at the domain edges. The active site is located in a groove and contains the classic catalytic triad of Ser, His and Asp. In the structure of the crystal soaked in diethyl p-nitrophenyl phosphate (DNP), the catalytic serine is covalently connected to a phosphonate moiety that clearly has only one ethyl group. This is the only example in the Protein Data Bank of a DNP-inhibited enzyme with covalently bound monoethylphosphate.

  20. From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review.

    PubMed

    Ramírez-Velasco, Lorena; Armendáriz-Ruiz, Mariana; Rodríguez-González, Jorge Alberto; Müller-Santos, Marcelo; Asaff-Torres, Ali; Mateos-Díaz, Juan Carlos

    2016-01-01

    Feruloyl esterases (FAEs) are a diverse group of hydrolases widely distributed in plants and microorganisms which catalyzes the cleavage and formation of ester bonds between plant cell wall polysaccharides and phenolic acids. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing highadded value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production, characterization and classification of FAEs, however only a few reports of suitable High Throughput Screening assays for this kind of enzymes have been reported. This review is focused on a concise but complete revision of classical to High Throughput Screening methods for FAEs, highlighting its advantages and disadvantages, and finally suggesting future perspectives for this important research field.

  1. Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937.

    PubMed

    Shevchik, V E; Hugouvieux-Cotte-Pattat, N

    1997-06-01

    Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of pae

  2. Bioassay technique using nonspecific esterase activities of Tetrahymena pyriformis for screening and assessing cytotoxicity of xenobiotics

    SciTech Connect

    Bogaerts, P.; Senaud, J.; Bohatier, J. |

    1998-08-01

    A simple and rapid test for screening and assessing the cytotoxicity of xenobiotics was developed with Tetrahymena pyriformis. The method estimates the activities of nonspecific esterases of a cell by concentrating within it a specific amount of fluorescence associated with fluorescein dye. The 2-h median effective concentration (EC50) values of 10 inorganic and eight organic substances are presented and compared to those of three other bioassays: the conventional T. pyriformis proliferation rate 9-h median inhibitory concentrations, the Microtox 30-min EC50s, and the Daphnia magna 4-methylumbelliferyl {beta}-D galactoside 1-h EC50s. A highly significant correlation was found between the results obtained with the fluorescein diacetate test and those obtained with the growth inhibition and Microtox tests. This in vivo enzymatic test showed high sensitivity to all compounds tested except Cr{sup 6+} and sodium dodecyl sulfate.

  3. Substituted 2-Aminopyrimidines Selective for α7-Nicotinic Acetylcholine Receptor Activation and Association with Acetylcholine Binding Proteins.

    PubMed

    Kaczanowska, Katarzyna; Camacho Hernandez, Gisela Andrea; Bendiks, Larissa; Kohs, Larissa; Cornejo-Bravo, Jose Manuel; Harel, Michal; Finn, M G; Taylor, Palmer

    2017-03-15

    Through studies with ligand binding to the acetylcholine binding protein (AChBP), we previously identified a series of 4,6-substituted 2-aminopyrimidines that associate with this soluble surrogate of the nicotinic acetylcholine receptor (nAChR) in a cooperative fashion, not seen for classical nicotinic agonists and antagonists. To examine receptor interactions of this structural family on ligand-gated ion channels, we employed HEK cells transfected with cDNAs encoding three requisite receptor subtypes: α7-nAChR, α4β2-nAChR, and a serotonin receptor (5-HT3AR), along with a fluorescent reporter. Initial screening of a series of over 50 newly characterized 2-aminopyrimidines with affinity for AChBP showed only two to be agonists on the α7-nAChR below 10 μM concentration. Their unique structural features were incorporated into design of a second subset of 2-aminopyrimidines yielding several congeners that elicited α7 activation with EC50 values of 70 nM and Kd values for AChBP in a similar range. Several compounds within this series exhibit specificity for the α7-nAChR, showing no activation or antagonism of α4β2-nAChR or 5-HT3AR at concentrations up to 10 μM, while others were weaker antagonists (or partial agonists) on these receptors. Analysis following cocrystallization of four ligand complexes with AChBP show binding at the subunit interface, but with an orientation or binding pose that differs from classical nicotinic agonists and antagonists and from the previously analyzed set of 2-aminopyrimidines that displayed distinct cooperative interactions with AChBP. Orientations of aromatic side chains of these complexes are distinctive, suggesting new modes of binding at the agonist-antagonist site and perhaps an allosteric action for heteromeric nAChRs.

  4. GDSL esterase/lipase genes in Brassica rapa L.: genome-wide identification and expression analysis.

    PubMed

    Dong, Xiangshu; Yi, Hankuil; Han, Ching-Tack; Nou, Ill-Sup; Hur, Yoonkang

    2016-04-01

    GDSL esterase/lipase proteins (GELPs), a very large subfamily of lipolytic enzymes, have been identified in microbes and many plants, but only a few have been characterized with respect to their roles in growth, development, and stress responses. In Brassica crops, as in many other species, genome-wide systematic analysis and functional studies of these genes are still lacking. As a first step to study their function in B. rapa ssp. pekinensis (Chinese cabbage), we comprehensively identified all GELP genes in the genome. We found a total of 121 Brassica rapa GDSL esterase/lipase protein genes (BrGELPs), forming three clades in the phylogenetic analysis (two major and one minor), with an asymmetrical chromosomal distribution. Most BrGELPs possess four strictly conserved residues (Ser-Gly-Asn-His) in four separate conserved regions, along with short conserved and clade-specific blocks, suggesting functional diversification of these proteins. Detailed expression profiling revealed that BrGELPs were expressed in various tissues, including floral organs, implying that BrGELPs play diverse roles in various tissues and during development. Ten percent of BrGELPs were specifically expressed in fertile buds, rather than male-sterile buds, implying their involvement in pollen development. Analyses of EXL6 (extracellular lipase 6) expression and its co-expressed genes in both B. rapa and Arabidopsis, as well as knockdown of this gene in Arabidopsis, revealed that this gene plays an important role in pollen development in both species. The data described in this study will facilitate future investigations of other BrGELP functions.

  5. Fundamental reaction mechanism and free energy profile for (-)-cocaine hydrolysis catalyzed by cocaine esterase.

    PubMed

    Liu, Junjun; Hamza, Adel; Zhan, Chang-Guo

    2009-08-26

    The fundamental reaction mechanism of cocaine esterase (CocE)-catalyzed hydrolysis of (-)-cocaine and the corresponding free energy profile have been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations. On the basis of the QM/MM-FE results, the entire hydrolysis reaction consists of four reaction steps, including the nucleophilic attack on the carbonyl carbon of (-)-cocaine benzoyl ester by the hydroxyl group of Ser117, dissociation of (-)-cocaine benzoyl ester, nucleophilic attack on the carbonyl carbon of (-)-cocaine benzoyl ester by water, and finally dissociation between the (-)-cocaine benzoyl group and Ser117 of CocE. The third reaction step involving the nucleophilic attack of a water molecule was found to be rate-determining, which is remarkably different from (-)-cocaine hydrolysis catalyzed by wild-type butyrylcholinesterase (BChE; where the formation of the prereactive BChE-(-)-cocaine complex is rate-determining) or its mutants containing Tyr332Gly or Tyr332Ala mutation (where the first chemical reaction step is rate-determining). Besides, the role of Asp259 in the catalytic triad of CocE does not follow the general concept of the "charge-relay system" for all serine esterases. The free energy barrier calculated for the rate-determining step of CocE-catalyzed hydrolysis of (-)-cocaine is 17.9 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 16.2 kcal/mol. In the present study, where many sodium ions are present, the effects of counterions are found to be significant in determining the free energy barrier. The finding of the significant effects of counterions on the free energy barrier may also be valuable in guiding future mechanistic studies on other charged enzymes.

  6. Influence of Randomly Inserted Feruloyl Esterase A on β-Glucosidase Activity in Trichoderma reesei.

    PubMed

    Hou, YunHua; Pan, Yang; Yan, MengJie; He, Huan; Yang, QinZheng; Zhong, YaoHua

    2017-02-24

    As a well-known industrial fungus for cellulase production, the strain RUT-C30 of Trichoderma reesei was selected to produce the feruloyl esterase A (FAEA) by a random integration protocol. The strong promoter of cellobiohydrolase 1 (cbh1) gene was used to drive the expression of FAEA. Using double-joint PCR protocol, Pcbh1-faeA-TtrpC expression cassette was successfully constructed and co-transformed into RUT C30 strain of T. reesei. One transformant with high feruloyl esterase yield (3.44 ± 0.16 IU/mL) was obtained through plate screening and named TrfaeA1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TrfaeA1 showed a distinct protein band appearing at the position of about 34 kDa, indicating that faeA gene has been successfully expressed in T. reesei. Compared with that in original RUT C30 strain, β-glucosidase production in transformant TrfaeA1 was significantly increased by about 86.4%, reaching 63.2 IU/mL due to the random insertion of faeA. Moreover, the total secretion protein and filter paper activities of the transformant TrfaeA1 were also improved by up to 5.5 and 4.3%, respectively. The present results indicated that the random insertion strategy could be an effective and feasible method to improve and optimize the cellulase system of filamentous fungi.

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

    PubMed

    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.

  8. The effect of depth of centrifuged synovial fluid on leukocyte esterase test for periprosthetic joint infection.

    PubMed

    Ruangsomboon, Pakpoom; Chinprasertsuk, Sriprapa; Khejonnit, Varanya; Chareancholvanich, Keerati

    2017-03-17

    Centrifugation of aspirated synovial fluid before leukocytes esterase (LE) testing for diagnosing periprosthetic joint infection (PJI) may make blood tinged specimens interpretable. We aimed to establish the proper sampling depth of centrifuged specimens for LE testing as one diagnostic criterion and also AS-D chloroacetate esterase (CAE) staining testing as an adjunctive tool. A definite PJI knee joint group and an aseptic primary total knee arthroplasty control group were studied quasi-experimentally (N = 46). At 2000 g for 15 minutes, 3 ml of synovial fluid was centrifuged. LE strip testing and median synovial WBC count were performed at 2, 4, and 6 mm depths. CAE staining test characterized LE particles. ROC curve, area under the curve, and significant differences were determined. The proper predictive depth to diagnose PJI was sought by forward stepwise logistic regression. All fresh blood-tinged specimens had uncertain interpretations. Centrifugation increased interpretability (55% to 100%). ROC curve and area under the curve at 2, 4, and 6 mm depths were 0.822, 0.804, and 0.786, respectively. The cut point of ++ to diagnose PJI was statistically significant (p < 0.05) at all depths. P-values of forward stepwise logistic regression at 2, 4, and 6 mm were 0.001, 0.752, and 0.756, respectively. CAE staining confirmed extracellular LE release by polymorphonuclear neutrophils (PMN). A specimen at < 2 mm from the surface of centrifuged synovial fluid at a grading of ++ or more for PJI diagnosis is proper for LE testing. CAE staining testing adjunctively characterizes LE particles and cell morphology. This article is protected by copyright. All rights reserved.

  9. Spruce budworm (Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning.

    PubMed

    Feng, Q L; Ladd, T R; Tomkins, B L; Sundaram, M; Sohi, S S; Retnakaran, A; Davey, K G; Palli, S R

    1999-02-25

    We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

  10. Tissue distribution, characterization and in vitro inhibition of B-esterases in the earwig Forficula auricularia.

    PubMed

    Malagnoux, Laure; Capowiez, Yvan; Rault, Magali

    2014-10-01

    Earwigs are important natural enemies of numerous pests in pome fruit orchards worldwide. Studying the effects of agricultural practices on these biological control agents is important for understanding its vulnerability in the field. The aim of this study was to characterize the B-esterase activities in the European earwig Forficula auricularia and to evaluate in vitro its sensitivity to organophosphate and carbamate pesticides. Acetylcholinesterase (AChE) activity was mainly measured with 1.5 mM acetylthiocholine as the substrate in the microsomal fraction of earwig heads (70% of total AChE activity). Carboxylesterase (CbE) activities were measured with three substrates [5 mM 4-nitrophenyl acetate (4-NPA), 1mM 4-nitrophenyl valerate (4-NPV), and 2 mM α-naphtyl acetate (α-NA)] to examine different isoenzymes, which were present mainly in the cytosolic fraction (about 70-88% of total activities) of all earwig tissues. CbE activity was higher than AChE activity, especially with α-NA, then 4-NPA and lastly 4-NPV. Chlorpyrifos-oxon an organophosphate, and carbaryl a carbamate pesticide, inhibited AChE and CbE activities in a concentration-dependent manner. Earwig CbE activities showed a stronger sensitivity to organophosphate than AChE, with the strongest effect for chlorpyrifos-oxon on male carboxylesterase activities. CbE and AChE showed about the same sensitivity to carbamate pesticides regardless of sex. These results suggest that B-type esterases in the European earwig F.auricularia are suitable biomarkers of pesticide exposure.

  11. Molecular Determinants for Competitive Inhibition of α4β2 Nicotinic Acetylcholine Receptors

    PubMed Central

    Carbone, Annalisa; García-Beltrán, Olimpo; Livingstone, Phil D.; Biggin, Philip C.; Cassels, Bruce K.; Wonnacott, Susan; Zapata-Torres, Gerald; Bermudez, Isabel

    2010-01-01

    The Erythrina alkaloids erysodine and dihydro-β-erythroidine (DHβE) are potent and selective competitive inhibitors of α4β2 nicotinic acetylcholine receptors (nAChRs), but little is known about the molecular determinants of the sensitivity of this receptor subtype to inhibition by this class of antagonists. We addressed this issue by examining the effects of DHβE and a range of aromatic Erythrina alkaloids on [3H]cytisine binding and receptor function in conjunction with homology models of the α4β2 nAChR, mutagenesis, and functional assays. The lactone group of DHβE and a hydroxyl group at position C-16 in aromatic Erythrina alkaloids were identified as major determinants of potency, which was decreased when the conserved residue Tyr126 in loop A of the α4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues α4Trp182 (loop B), α4Tyr230 (loop C), and β2Trp82 (loop D) and the nonconserved β2Thr84; however, only α4Trp182 was predicted to contact bound antagonist, suggesting α4Tyr230, β2Trp82, and β2Thr84 contribute allosterically to the closed state elicited by bound antagonist. In addition, homology modeling predicted strong ionic interactions between the ammonium center of the Erythrina alkaloids and β2Asp196, leading to the uncapping of loop C. Consistent with this, β2D196A abolished sensitivity to inhibition by DHβE or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with β2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhibition by DHβE or erysodine, which highlights β2Asp196 as a major determinant of the receptor selectivity of Erythrina alkaloids. PMID:20547737

  12. Calcium responses induced by acetylcholine in submucosal arterioles of the guinea-pig small intestine

    PubMed Central

    Fukuta, Hiroyasu; Hashitani, Hikaru; Yamamoto, Yoshimichi; Suzuki, Hikaru

    1999-01-01

    Calcium responses induced by brief stimulation with acetylcholine (ACh) were assessed from the fluorescence changes in fura-2 loaded submucosal arterioles of the guinea-pig small intestine. Initially, 1–1.5 h after loading with fura-2 (fresh tissues), ACh increased [Ca2+]i in a concentration-dependent manner. This response diminished with time, and finally disappeared in 2–3 h (old tissues). Ba2+ elevated [Ca2+]i to a similar extent in both fresh and old tissues. ACh further increased the Ba2+-elevated [Ca2+]i in fresh tissues, but reduced it in old tissues. Responses were not affected by either indomethacin or nitroarginine. In fresh mesenteric arteries, mechanical removal of endothelial cells abolished the ACh-induced increase in [Ca2+]i, with no alteration of [Ca2+]i at rest and during elevation with Ba2+. In the presence of indomethacin and nitroarginine, high-K+ solution elevated [Ca2+]i in both fresh and old tissues. Subsequent addition of ACh further increased [Ca2+]i in fresh tissues without changing it in old tissues. Proadifen, an inhibitor of the enzyme cytochrome P450 mono-oxygenase, inhibited the ACh-induced changes in [Ca2+]i in both fresh and Ba2+-stimulated old tissues. It also inhibited the ACh-induced hyperpolarization. In fresh tissues, the ACh-induced Ca2+ response was not changed by apamin, charybdotoxin (CTX), 4-aminopyridine (4-AP) or glibenclamide. In old tissues in which [Ca2+]i had previously been elevated with Ba2+, the ACh-induced Ca2+ response was inhibited by CTX but not by apamin, 4-AP or glibenclamide. It is concluded that in submucosal arterioles, ACh elevates endothelial [Ca2+]i and reduces muscular [Ca2+]i, probably through the hyperpolarization of endothelial or smooth muscle membrane by activating CTX-sensitive K+ channels. PMID:10050015

  13. Endogenous Inhibition of the Trigeminally Evoked Neurotransmission to Cardiac Vagal Neurons by Muscarinic Acetylcholine Receptors

    PubMed Central

    Gorini, C.; Philbin, K.; Bateman, R.

    2010-01-01

    Stimulation of the nasal mucosa by airborne irritants or water evokes a pronounced bradycardia accompanied by peripheral vasoconstriction and apnea. The dive response, which includes the trigeminocardiac reflex, is among the most powerful autonomic responses. These responses slow the heart rate and reduce myocardial oxygen consumption. Although normally cardioprotective, exaggeration of this reflex can be detrimental and has been implicated in cardiorespiratory diseases, including sudden infant death syndrome (SIDS). An essential component of the diving response and trigeminocardiac reflex is activation of the parasympathetic cardiac vagal neurons (CVNs) in the nucleus ambiguus that control heart rate. This study examined the involvement of cholinergic receptors in trigeminally evoked excitatory postsynaptic currents in CVNs in an in vitro preparation from rats. CVNs were identified using a retrograde tracer injected into the fat pads at the base of the heart. Application of the acetylcholinesterase inhibitor neostigmine significantly decreased the amplitude of glutamatergic neurotransmission to CVNs on stimulation of trigeminal fibers. Whereas nicotine did not have any effect on the glutamatergic responses, the muscarinic acetylcholine receptor (mAChR) agonist bethanechol significantly decreased the excitatory neurotransmission. Atropine, an mAChR antagonist, facilitated these responses indicating this trigeminally evoked brain stem pathway in vitro is endogenously inhibited by mAChRs. Tropicamide, an m4 mAChR antagonist, prevented the inhibitory action of the muscarinic agonist bethanechol. These results indicate that the glutamatergic synaptic neurotransmission in the trigeminally evoked pathway to CVNs is endogenously inhibited in vitro by m4 mAChRs. PMID:20719927

  14. Depression of miniature endplate potential frequency by acetylcholine and its analogues in frog.

    PubMed Central

    Nikolsky, E. E.; Bukharaeva, E. A.; Strunsky, E. G.; Vyskocil, F.

    1991-01-01

    1. Acetylcholine (ACh), 7.5 x 10(-5) M, and carbachol, 5 x 10(-6) M (CCh) depressed the frequency of miniature endplate potentials (m.e.p.ps) in the frog (Rana temporaria) sartorius neuromuscular junction with active acetylcholinesterase to about 50-55% of the controls. 2. A similar depression was produced by the nicotinic agonists, nicotine, suberyldicholine and tetramethylammonium. 3. The muscarinic agonists, oxotremorine, methylfurmethide and methacholine were without effect on m.e.p.p. frequency. The muscarinic antagonist, atropine and the nicotinic antagonist, (+)-tubocurarine, had no effect on the depression of m.e.p.p. frequency evoked by CCh. 4. The ganglionic blockers, benzhexonium and IEM-1119, were also without effect on the CCh-evoked depression of m.e.p.p. frequency. 5. Pretreatment of muscles with anticholinesterases did not prevent the CCh-induced drop in m.e.p.p. frequency. 6. The effect of CCh was proportionally the same as in the controls in preparations where the m.e.p.p. frequency was changed by elevation of K+ and in the presence of theophylline, noradrenaline, dibutyryl adenosine 3':5'-cyclic monophosphate (db cyclic AMP) and db cyclic GMP. 7. An inhibitor of Na+,K(+)-ATPase, ouabain, 5 x 10(-5) mol l-1, prevented or reversed the depression of m.e.p.p. frequency by CCh. However, the depression was present in a nominally K(+)-free medium. Insulin and adrenaline, which are considered to be Na+,K(+)-ATPase activators, were without effect on depression of m.e.p.p. frequency. 8. The depression of m.e.p.p. frequency by 5 x 10(-6) M CCh was the same at temperatures between 5 and 30 degrees C with a Q10 near to 1.0.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1667283

  15. Luminal acetylcholine does not affect the activity of the CFTR in tracheal epithelia of pigs.

    PubMed

    Dittrich, Nikolaus P; Kummer, Wolfgang; Clauss, Wolfgang G; Fronius, Martin

    2015-11-01

    Fluid homeostasis mediated by the airway epithelium is required for proper lung function, and the CFTR (cystic fibrosis transmembrane conductance regulator) Cl(-) channel is crucial for these processes. Luminal acetylcholine (ACh) acts as an auto-/paracrine mediator to activate Cl(-) channels in airway epithelia and evidence exists showing that nicotinic ACh receptors activate CFTR in murine airway epithelia. The present study investigated whether or not luminal ACh regulates CFTR activity in airway epithelia of pigs, an emerging model for investigations of human airway disease and cystic fibrosis (CF) in particular. Transepithelial ion currents of freshly dissected pig tracheal preparations were measured with Ussing chambers. Application of luminal ACh (100 μM) induced an increase of the short-circuit current (I(SC)). The ACh effect was mimicked by muscarine and pilocarpine (100 μM each) and was sensitive to muscarinic receptor antagonists (atropine, 4-DAMP, pirenzepine). No changes of the I(SC) were observed by nicotine (100 μM) and ACh responses were not affected by nicotine or mecamylamine (25 μM). Luminal application of IBMX (I, 100 μM) and forskolin (F, 10 μM), increase the I(SC) and the I/F-induced current were decreased by the CFTR inhibitor GlyH-101 (GlyH, 50 μM) indicating increased CFTR activity by I/F. In contrast, GlyH did not affect the ACh-induced current, indicating that the ACh response does not involve the activation of the CFTR. Results from this study suggest that luminal ACh does not regulate the activity of the CFTR in tracheal epithelia of pigs which opposes observation from studies using mice airway epithelium.

  16. Rabies virus interaction with various cell lines is independent of the acetylcholine receptor.

    PubMed

    Reagan, K J; Wunner, W H

    1985-01-01

    Rabies virus infects most cells in vitro. The presence of the nicotinic acetylcholine receptor on the plasma membrane of various cell lines is not an obligate factor for rabies virus susceptibility of those cells.

  17. Cyclic nucleotides of canine antral smooth muscle. Effects of acetylcholine, catecholamines and gastrin.

    PubMed

    Baur, S; Grant, B; Wooton, J

    1981-01-07

    1. The effects of acetylcholine, catecholamines and gastrin on the intracellular content of cyclic AMP and cyclic GMP in antral circular muscle have been determined. 2. Acetylcholine results in a significant but transient increase in intracellular cyclic GMP. 3. Isoproterenol and norepinephrine increase intracellular cyclic AMP. Based on half-maximal effective doses, isoproterenol is 2.7-times more effective than norepinephrine. The increase in intracellular cyclic AMP by both agents is inhibited by propranolol but not phentolamine, indicating that both agents act on the muscle cell by a beta-receptor-coupled mechanism. 4. Gastrin has no demonstrable effect on either cyclic AMP or cyclic GMP. This suggests that while gastrin and acetylcholine can produce a like myoelectric response in the muscle cell, the action of gastrin is mediated by a separate receptor, presumably on the muscle cell, and not by a release of acetylcholine.

  18. Effect of opioid peptides on electrically evoked acetylcholine release from Torpedo electromotor neurons.

    PubMed

    Oron, L; Sarne, Y; Michaelson, D M

    1991-04-29

    The opioid peptide dynorphin A(1-8) (1 micron) increased acetylcholine release from the Torpedo electric organ by approximately twofold. This effect was reversed by the opiate antagonist naloxone. The effect of Dyn A(1-8) on acetylcholine release was found to vary in magnitude with the seasons of the year, with maximal enhancement being observed in the summer and none in winter. Dynorphin B, methionine-enkephalin and leucine-enkephalin also increased acetylcholine release and showed similar seasonal variations. These findings suggest that acetylcholine release from Torpedo electromotor neurons is regulated by opiate receptors. The physiological significance of these observations is discussed in view of the previous findings that the Torpedo neurons contain an endogenous enkephalin-like peptide.

  19. Nonenzymatic all-solid-state coated wire electrode for acetylcholine determination in vitro.

    PubMed

    He, Cheng; Wang, Zhan; Wang, You; Hu, Ruifen; Li, Guang

    2016-11-15

    A nonenzymatic all-solid-state coated wire acetylcholine electrode was investigated. Poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT/PSS) as conducting polymer was coated on one end of a gold wire (0.5mm in diameter). The acetylcholine selective membrane containing heptakis(2,3,6-tri-Ο-methyl)-β-cyclodextrin as an ionophore covered the conducting polymer layer. The electrode could work stably in a pH range of 6.5-8.5 and a temperature range of 15-40°C. It covered an acetylcholine concentration range of 10(-5)-10(-1)M with a slope of 54.04±1.70mV/decade, while detection limit was 5.69±1.06µM. The selectivity, dynamic response, reproducibility and stability were evaluated. The electrode could work properly in the rat brain homogenate to detect different concentrations of acetylcholine.

  20. Notexin preferentially inhibits the release of newly synthesized acetylcholine from rat brain synaptosomal fractions

    SciTech Connect

    Gundersen, C.B.; Jenden, D.J.

    1981-01-01

    An investigation was made of the effects of the snake venom neurotoxin, notexin, on acetylcholine turnover in rat brain P2 fractions using a gas chromatographic mass spectrometric assay for acetylcholine and choline. In contrast to earlier reports, we found a stimulation of the uptake and acetylation of labeled choline by toxin-treated P2 fractions. More significantly, notexin inhibited the release of this newly synthesized transmitter. These effects were found to be dependent on the dose of the toxin and the time of exposure of the P2 fraction to notexin. Longer exposure to notexin or experiments involving resuspension of notexin-treated P2 fractions appeared to result in considerable lysis of the transmitter-containing particles. Thus, notexin may alter acetylcholine compartmentation in the nerve ending and thereby affect acetylcholine synthesis.

  1. Microtransplantation of acetylcholine receptors from normal or denervated rat skeletal muscles to frog oocytes

    PubMed Central

    Bernareggi, Annalisa; Reyes-Ruiz, Jorge Mauricio; Lorenzon, Paola; Ruzzier, Fabio; Miledi, Ricardo

    2011-01-01

    Cell membranes, carrying neurotransmitter receptors and ion channels, can be ‘microtransplanted’ into frog oocytes. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have, still embedded in their natural lipid environment. This approach has been previously demonstrated to be very useful to study neurotransmitter receptors and ion channels contained in cell membranes isolated from human brains. Here, we examined the possibility of using the microtransplantation method to study acetylcholine receptors from normal and denervated rat skeletal muscles. We found that the muscle membranes, carrying their fetal or adult acetylcholine receptor isoforms, could be efficiently microtransplanted to the oocyte membrane, making the oocytes become sensitive to acetylcholine. These results show that oocytes injected with skeletal muscle membranes efficiently incorporate functional acetylcholine receptors, thus making the microtransplantation approach a valuable tool to further investigate receptors and ion channels of human muscle diseases. PMID:21224230

  2. A role for acetylcholine receptors in the fusion of chick myoblasts

    PubMed Central

    1988-01-01

    The role of acetylcholine receptors in the control of chick myoblast fusion in culture has been explored. Spontaneous fusion of myoblasts was inhibited by the nicotinic acetylcholine receptor antagonists alpha- bungarotoxin, Naja naja toxin and monoclonal antibody mcAb 5.5. The muscarinic antagonists QNB and n-methyl scopolamine were without effect. Atropine had no effect below 1 microM, where it blocks muscarinic receptors; at higher concentrations, when it blocks nicotinic receptors also, atropine inhibited myoblast fusion. The inhibitions imposed by acetylcholine receptor antagonists lasted for approximately 12 h; fusion stimulated by other endogenous substances then took over. The inhibition was limited to myoblast fusion. The increases in cell number, DNA content, the level of creatine phosphokinase activity (both total and muscle-specific isozyme) and the appearance of heavy chain myosin, which accompany muscle differentiation, followed a normal time course. Pre-fusion myoblasts, fusing myoblasts, and young myotubes specifically bound labeled alpha- bungarotoxin, indicating the presence of acetylcholine receptors. The nicotinic acetylcholine receptor agonist, carbachol, induced uptake of [14C]Guanidinium through the acetylcholine receptor. Myoblasts, aligned myoblasts and young myotubes expressed the synthetic enzyme Choline acetyltransferase and stained positively with antibodies against acetylcholine. The appearance of ChAT activity in myogenic cultures was prevented by treatment with BUDR; nonmyogenic cells in the cultures expressed ChAT at a level which was too low to account for the activity in myogenic cultures. We conclude that activation of the nicotinic acetylcholine receptor is part of the mechanism controlling spontaneous myoblast fusion and that myoblasts synthesize an endogenous, fusion- inducing agent that activates the nicotinic ACh receptor. PMID:3372592

  3. Role of dopamine receptor and muscarinic acetylcholine receptor blockade in the antiapomorphine action of neuroleptics

    SciTech Connect

    Zharkovskii, A.M.; Langel, Yu.L.; Chereshka, K.S.; Zharkovskaya, T.A.

    1987-08-01

    The authors analyze the role of dopamine and muscarinic acetylcholine receptor blocking components in the antistereotypic action of neuroleptics with different chemical structure. To determine dopamine-blocking activity in vitro, binding of /sup 3/H-spiperone with membranes of the rat striatum was measured. To study the blocking action of the substances on muscarinic acetylcholine receptors, binding of /sup 3/H-quinuclidinyl benzylate with brain membranes was chosen.

  4. Acetylcholine test in patients with angina pectoris and normal coronary angiography

    NASA Astrophysics Data System (ADS)

    Barbieri, Enrico; Destro, Gianni; Oliva, Massimo; Zardini, Piero

    1994-02-01

    Angina pectoris with normal coronary artery on the coronary angiography is an intriguing issue. Intracoronary infusion of acetylcholine has recently been used to test the integrity of endothelial cells. We studied 16 patients with this syndrome. A relationship has been found between the acetylcholine test and the exercise stress test in normotensive patients. The presence of hypertension makes the evaluation of the test more unpredictable, probably because of the damage on the endothelial cells related to systemic hypertension.

  5. Subpopulations of rat dorsal root ganglion neurons express active vesicular acetylcholine transporter.

    PubMed

    Tata, Ada Maria; De Stefano, M Egle; Tomassy, Giulio Srubek; Vilaró, M Teresa; Levey, Allan I; Biagioni, Stefano

    2004-01-15

    The vesicular acetylcholine transporter (VAChT) is a transmembrane protein required, in cholinergic neurons, for selective storage of acetylcholine into synaptic vesicles. Although dorsal root ganglion (DRG) neurons utilize neuropeptides and amino acids for neurotransmission, we have previously demonstrated the presence of a cholinergic system. To investigate whether, in sensory neurons, the vesicular accumulation of acetylcholine relies on the same mechanisms active in classical cholinergic neurons, we investigated VAChT presence, subcellular distribution, and activity. RT-PCR and Western blot analysis demonstrated the presence of VAChT mRNA and protein product in DRG neurons and in the striatum and cortex, used as positive controls. Moreover, in situ hybridization and immunocytochemistry showed VAChT staining located mainly in the medium/large-sized subpopulation of the sensory neurons. A few small neurons were also faintly labeled by immunocytochemistry. In the electron microscope, immunolabeling was associated with vesicle-like elements distributed in the neuronal cytoplasm and in both myelinated and unmyelinated intraganglionic nerve fibers. Finally, [(3)H]acetylcholine active transport, evaluated either in the presence or in the absence of ATP, also demonstrated that, as previously reported, the uptake of acetylcholine by VAChT is ATP dependent. This study suggests that DRG neurons not only are able to synthesize and degrade ACh and to convey cholinergic stimuli but also are capable of accumulating and, possibly, releasing acetylcholine by the same mechanism used by the better known cholinergic neurons.

  6. Detection of basal and potassium-evoked acetylcholine release from embryonic DRG explants.

    PubMed

    Bernardini, Nadia; Tomassy, Giulio Srubek; Tata, Ada Maria; Augusti-Tocco, Gabriella; Biagioni, Stefano

    2004-03-01

    Spontaneous and potassium-induced acetylcholine release from embryonic (E12 and E18) chick dorsal root ganglia explants at 3 and 7 days in culture was investigated using a chemiluminescent procedure. A basal release ranging from 2.4 to 13.8 pm/ganglion/5 min was detected. Potassium application always induced a significant increase over the basal release. The acetylcholine levels measured in E12 explants were 6.3 and 38.4 pm/ganglion/5 min at 3 and 7 days in culture, respectively, while in E18 explant cultures they were 10.7 and 15.5 pm/ganglion/5 min. In experiments performed in the absence of extracellular Ca2+ ions, acetylcholine release, both basal and potassium-induced, was abolished and it was reduced by cholinergic antagonists. A morphometric analysis of explant fibre length suggested that acetylcholine release was directly correlated to neurite extension. Moreover, treatment of E12 dorsal root ganglion-dissociated cell cultures with carbachol as cholinergic receptor agonist was shown to induce a higher neurite outgrowth compared with untreated cultures. The concomitant treatment with carbachol and the antagonists at muscarinic receptors atropine and at nicotinic receptors mecamylamine counteracted the increase in fibre outgrowth. Although the present data have not established whether acetylcholine is released by neurones or glial cells, these observations provide the first evidence of a regulated release of acetylcholine in dorsal root ganglia.

  7. Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells.

    PubMed

    Arai, Yuki; Ishii, Hirotaka; Kobayashi, Makito; Ozawa, Hitoshi

    2017-03-01

    GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca(2+) influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

  8. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  9. Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

    PubMed Central

    Leis, Benedikt; Angelov, Angel; Mientus, Markus; Li, Haijuan; Pham, Vu T. T.; Lauinger, Benjamin; Bongen, Patrick; Pietruszka, Jörg; Gonçalves, Luís G.; Santos, Helena; Liebl, Wolfgang

    2015-01-01

    Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and

  10. Functional Characterization of a Robust Marine Microbial Esterase and Its Utilization in the Stereo-Selective Preparation of Ethyl (S)-3-Hydroxybutyrate.

    PubMed

    Wang, Yilong; Zhang, Yun; Hu, Yunfeng

    2016-11-01

    One novel microbial esterase PHE21 was cloned from the genome of Pseudomonas oryzihabitans HUP022 identified from the deep sea of the Western Pacific. PHE21 was heterologously expressed and functionally characterized to be a robust esterase which behaved high resistance to various metal ions, organic solvents, surfactants, and NaCl. Despite the fact that the two enantiomers of ethyl 3-hydroxybutyrate were hard to be enzymatically resolved before, we successfully resolved racemic ethyl 3-hydroxybutyrate through direct hydrolysis reactions and generated chiral ethyl (S)-3-hydroxybutyrate using esterase PHE21. After process optimization, the enantiomeric excess, the conversion rate, and the yield of desired product ethyl (S)-3-hydroxybutyrate could reach 99, 65, and 87 %, respectively. PHE21 is a novel marine microbial esterase with great potential in asymmetric synthesis as well as in other industries.

  11. Histochemical studies on genetical control of hormonal enzyme inducibility in the mouse. I. Non-specific esterase activity and regional histology of the epididymis.

    PubMed Central

    Blecher, S R; Kirkeby, S

    1978-01-01

    As a base line for future cell genetical studies the authors record the distribution of non-specific esterase reaction in the various histologically distinguishable cell types of the mouse epididymis. The findings are correlated with previous descriptions of the lobar structure of the organ. Assuming the sequence of lobes of the head to be as implied in these classical descriptions, the esterase activity of the epithelial cells gradates between strong to weak several times along the length of the epididymal duct. The relationship of the lobes to each other, as seen in transverse sections, is described. Methodological studies using different fixatives indicate that apparent similarity of esterase reaction at different sites may camouflage an underlying difference in the nature of the esterases at these sites. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:564339

  12. Acetylcholine-induced K+ currents in smooth muscle cells of intact rat small arteries.

    PubMed Central

    Weidelt, T; Boldt, W; Markwardt, F

    1997-01-01

    1. The mechanism of the sustained acetylcholine-induced endothelium-dependent hyperpolarization (EDH) in intact rat small mesenteric arteries prestimulated with noradrenaline (10(-6) M) was investigated by means of the single microelectrode voltage-clamp method. 2. The vascular smooth muscle cells (VSMCs) in this preparation are poorly or even not coupled for the reasons that: (1) the mean input resistance Rlnp of the clamped vascular smooth muscle increases from 120 M omega under control conditions to 440 M omega after application of K+ channel blocking drugs, (2) the voltage relaxation after injection of hyperpolarizing currents has a monoexponential time course and is linearly dependent on Rlnp, and (3) voltage steps induced by current-clamp steps are not transferred to locations in the vascular musculature 120 microns apart from the current injecting microelectrode. 3. Sustained (> 5 min) application of ACh (10(-5) M) hyperpolarized the VSMCs by induction of a hyperpolarizing current. This effect was completely blocked by the inhibitor of the nitric oxide (NO) synthase L-NAME (10(-3) M) but not by the inhibitor of the soluble guanylate cyclase (sGCl) Methylene Blue (MB, 10(-4) M). 4. Application of the NO donor sodium nitroprusside (SNP, 10(-6) M) for more than 5 min mimicked the induction of the endothelium-dependent hyperpolarizing current in vessels with destroyed endothelium. The reversal potential of this current is dependent on the extracellular K+ concentration. The effect of SNP could also not be blocked by MB. 5. The blockers of ATP-dependent and Ca(2+)-dependent K+ channels, glibenclamide (Glb, 10(-5) M) and charybdotoxin (CTX, 5 x 10(-8) M), respectively, blocked a hyperpolarizing current in the VSMCs similar to the ACh- or SNP-induced current. 6. The isolated application of either Glb or CTX did not block the activation of the hyperpolarizing current by SNP. Only the combined administration of Glb and CTX blocked the SNP-induced current completely

  13. Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

    PubMed Central

    1990-01-01

    The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin- treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits

  14. Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase

    PubMed Central

    Nedrud, David M.; Lin, Hui; Lopez, Gilsinia; Padhi, Santosh K.; Legatt, Graig A.

    2014-01-01

    Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min−1 for hydrolysis of p-nitrophenyl acetate. Adding a third substitution – Glu79His – increased esterase activity more than tenfold to kcat ~ 1.6 min−1. The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at

  15. Synergism between insecticides permethrin and propoxur occurs through activation of presynaptic muscarinic negative feedback of acetylcholine release in the insect central nervous system.

    PubMed

    Corbel, Vincent; Stankiewicz, Maria; Bonnet, Julien; Grolleau, Françoise; Hougard, Jean Marc; Lapied, Bruno

    2006-07-01

    Although synergism between pesticides has been widely documented, the physiological mechanisms by which an insecticide synergizes another remains unclear. Toxicological and electrophysiological studies were carried out on two susceptible pest species (the mosquito Culex quinquefasciatus and the cockroach Periplaneta americana) to understand better the physiological process involved in pyrethroid and carbamate interactions. Larval bioassays were conducted with the susceptible reference strain SLAB of C. quinquefasciatus to assess the implication of multi-function oxidases and non-specific esterases in insecticide detoxification and synergism. Results showed that the general theory of synergism (competition between pesticides for a common detoxification enzyme) was unlikely to occur in the SLAB strain since the level of synergy recorded between permethrin and propoxur was unchanged in the presence of piperonyl butoxide and tribufos, two inhibitors of oxidases and esterases, respectively (synergism ratios were similar with and without synergists). We also showed that addition of a sub-lethal concentration of nicotine significantly increased the toxicity of permethrin and propoxur at the lower range of the dose-mortality regression lines, suggesting the manifestation of important physiological disruptions at synaptic level. The effects of both permethrin and propoxur were studied on the cercal-afferent giant-interneuron synapses in the terminal abdominal ganglion of the cockroach P. americana using the single-fibre oil-gap method. We demonstrated that permethrin and propoxur increased drastically the ACh concentration within the synaptic cleft, which thereby stimulated a negative feedback of ACh release. Atropine, a muscarinic receptor antagonist, reversed the effect of permethrin and propoxur mixtures. This demonstrates the implication of the presynaptic muscarinic receptors in the negative feedback regulation process and in synergism. Based on these findings, we

  16. The effects of substance P and acetylcholine on human tenocyte proliferation converge mechanistically via TGF-β1

    PubMed Central

    Fong, Gloria; Backman, Ludvig J.; Alfredson, Håkan; Scott, Alex; Danielson, Patrik

    2017-01-01

    Previous in vitro studies on human tendon cells (tenocytes) have demonstrated that the exogenous administration of substance P (SP) and acetylcholine (ACh) independently result in tenocyte proliferation, which is a prominent feature of tendinosis. Interestingly, the possible link between SP and ACh has not yet been explored in human tenocytes. Recent studies in other cell types demonstrate that both SP and ACh independently upregulate TGF-β1 expression via their respective receptors, the neurokinin 1 receptor (NK-1R) and muscarinic ACh receptors (mAChRs). Furthermore, TGF-β1 has been shown to downregulate NK-1R expression in human keratocytes. The aim of this study was to examine if TGF-β1 is the intermediary player involved in mediating the proliferative pathway shared by SP and ACh in human tenocytes. The results showed that exogenous administration of SP and ACh both caused significant upregulation of TGF-β1 at the mRNA and protein levels. Exposing cells to TGF-β1 resulted in increased cell viability of tenocytes, which was blocked in the presence of the TGFβRI/II kinase inhibitor. In addition, the proliferative effects of SP and ACh on tenocytes were reduced by the TGFβRI/II kinase inhibitor; this supports the hypothesis that the proliferative effects of these signal substances are mediated via the TGF-β axis. Furthermore, exogenous TGF-β1 downregulated NK-1R and mAChRs expression at both the mRNA and protein levels, and these effects were negated by simultaneous exposure to the TGFβRI/II kinase inhibitor, suggesting a negative feedback loop. In conclusion, the results indicate that TGF-β1 is the intermediary player through which the proliferative actions of both SP and ACh converge mechanistically. PMID:28301610

  17. Nitric oxide synthase inhibitors do not alter functional hyperemia in canine skeletal muscle.

    PubMed

    Barclay, J K; Woodley, N E

    1994-09-01

    To test the hypothesis that endothelium-derived products contribute to functional hyperemia in skeletal muscle, we infused nitric oxide synthase inhibitors, either 200 microM N omega-nitro-L-arginine (NNA) (N = 4) or 1 mM N gamma-monomethyl-L-arginine (NMMA) (N = 4), before and during 6 min of 4 Hz stimulation of canine gastrocnemius in situ. We infused saline (N = 4) as a control. NNA significantly decreased steady-level resting flow by 3.8 +/- 0.4 mL.kg-1.s-1. The increase in flow from rest to 5 min of stimulation was not changed by the nitric oxide synthase inhibitors. We also stimulated muscles for 60 min either with saline infusion (N = 4) or with the infusion of saline during the first 15 min and NNA for the remaining 45 min (n = 4). There was no difference in the flow during contractions. To clarify the effect of these inhibitors on canine vessels, we challenged rings of canine femoral artery with and without endothelium with acetylcholine and bradykinin (both 1 microM) before and after the addition of NNA and NMMA (both 10 microM). The nitric oxide synthase inhibitors decreased the relaxation accompanying acetylcholine. Both inhibitors caused only endothelium-intact rings to contract. Thus, the presence of a nitric oxide synthase inhibitor identified an endothelium-dependent contribution to the regulation of blood flow to skeletal muscle at rest but had no effect on functional hyperemia.

  18. Cloning, expression and characterization of a novel cold-active and organic solvent-tolerant esterase from Monascus ruber M7.

    PubMed

    Guo, Hailun; Zhang, Yan; Shao, Yanchun; Chen, Wanping; Chen, Fusheng; Li, Mu

    2016-07-01

    Cold active esterases are a class of important biocatalysts that exhibit high activity at low temperatures. In this study, a search for putative cold-active esterase encoding genes from Monascus ruber M7 was performed. A cold-active esterase, named Lip10, was isolated, cloned, purified, and characterized. Amino acid sequence analysis reveals that Lip10 contained a conserved sequence motif Gly(173)-Xaa-Ser(175)-Xaa-Gly(177) that is also present in the majority of esterases and lipases. Phylogenetic analysis indicated that Lip10 was a novel microbial esterase. The lip10 gene was cloned and heterologously expressed in Escherichia coli BL21(DE3), resulting in the expression of an active and soluble protein that constituted 40 % of the total cell protein content. Lip10 maintained almost 50 % of its maximal activity at 4-10 °C, with optimal activity at 40 °C. Furthermore, Lip10 retained 184-216 % of its original activity, after incubation in 50 % (v/v) hydrophobic organic solvents for 24 h. The enzyme also exhibited high activity under alkaline conditions and good tolerance to metal ions in the reaction mixture. These results indicate that Lip10 may have potential uses in chemical synthesis and food processing industrial applications as an esterase.

  19. Looking below the surface of nicotinic acetylcholine receptors

    PubMed Central

    Stokes, Clare; Treinin, Millet; Papke, Roger L.

    2015-01-01

    The amino acid sequences of nicotinic acetylcholine receptors (nAChRs) from diverse species can be compared across extracellular, transmembrane, and intracellular domains. The intracellular domains are most divergent among subtypes, yet relatively consistent among species. The diversity indicates that each nAChR subtype possesses a unique language for communication with its host cell. The conservation across species also suggests that the intracellular domains may play defining functional roles for each subtype. Secondary structure prediction indicates two relatively conserved alpha helices within the intracellular domains of all nAChRs. Among all subtypes, the intracellular domain of α7 nAChR is one of the most-well conserved, and α7 nAChRs have effects in non-neuronal cells independent of generating ion currents, making it likely that the α7 intracellular domain directly mediates signal transduction. There are potential phosphorylation and protein binding sites in the α7 intracellular domain, which are conserved and may be the basis for α7-mediated signal transduction. PMID:26067101

  20. Vasoactive intestinal polypeptide provokes acetylcholine release from the myenteric plexus

    SciTech Connect

    Kusunoki, M.; Tsai, L.H.; Taniyama, K.; Tanaka, C.

    1986-07-01

    Effects of vasoactive intestinal polypeptide (VIP) on the release of acetylcholine (ACh) from longitudinal muscle strips with myenteric plexus (LM) preparations were examined in the guinea pig small intestine. VIP (10 to 10 W M) induced a concentration-dependent contraction of LM preparation. The VIP-induced contractions seem to be related to three components, the scopolamine-sensitive, the scopolamine-insensitive, the tetrodotoxin-sensitive, and the tetrodotoxin-insensitive contractions. VIP (10 to 10 W M) induced a concentration-dependent increase in the release of (TH)ACh from LM preparations preloaded with (TH)choline. The VIP-evoked (TH)ACh release was inhibited by removal of CaS from the perfusion medium and by treatment with tetrodotoxin but not by scopolamine and hexamethonium. The spontaneous and VIP-evoked (TH)ACh release was not affected by phentolamine, propranolol, methysergide, diphenhydramine, cimetidine, bicuculline, or (D-ProS, D-Trp/sup 7,9/)substance P. The result demonstrates that VIP induces contractions of longitudinal smooth muscle directly and indirectly by the stimulation of both cholinergic neurons and noncholinergic excitatory neurons.

  1. Regulation of hippocampal inhibitory circuits by nicotinic acetylcholine receptors

    PubMed Central

    Griguoli, Marilena; Cherubini, Enrico

    2012-01-01

    The hippocampal network comprises a large variety of locally connected GABAergic interneurons exerting a powerful control on network excitability and which are responsible for the oscillatory behaviour crucial for information processing. GABAergic interneurons receive an important cholinergic innervation from the medial septum-diagonal band complex of the basal forebrain and are endowed with a variety of muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) that regulate their activity. Deficits in the cholinergic system lead to the impairment of high cognitive functions, which are particularly relevant in neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases as well as in schizophrenia. Here, we highlight some recent advances in the mechanisms by which cholinergic signalling via nAChRs regulates local inhibitory circuits in the hippocampus, early in postnatal life and in adulthood. We also discuss recent findings concerning the functional role of nAChRs in controlling short- and long-term modifications of synaptic efficacy. Insights into these processes may provide new targets for the therapeutic control of pathological conditions associated with cholinergic dysfunctions. PMID:22124144

  2. Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica

    PubMed Central

    Du, Nana; Liu, Yanfang; Zhang, Xiuli; Wang, Jixia; Zhao, Jianqiang; He, Jian; Zhou, Han; Mei, Lijuan; Liang, Xinmiao

    2017-01-01

    Scopolia tangutica (S. tangutica) is a traditional Chinese medicinal plant used for antispasmodics, anesthesia, analgesia and sedation. Its pharmacological activities are mostly associated with the antagonistic activity at muscarinic acetylcholine receptors (mAchRs) of several known alkaloids such as atropine and scopolamine. With our recent identification of four hydroxycinnamic acid amides from S. tangutica, we hypothesized that this plant may contain previously unidentified alkaloids that may also contribute to its in vivo effect. Herein, we used a bioassay-guided multi-dimension separation strategy to discover novel mAchR antagonists from S. tangutica. The core of this approach is to use label-free cell phenotypic assay to first identify active fractions, and then to guide purification of active ligands. Besides four tropanes and six cinnamic acid amides that have been previously isolated from S. tangutica, we recently identified two new tropanes, one new cinnamic acid amide, and nine other compounds. Six tropane compounds purified from S. tangutica for the first time were confirmed to be competitive antagonists of muscarinic receptor 3 (M3), including the two new ones 8 and 12 with IC50 values of 1.97 μM and 4.47 μM, respectively. Furthermore, the cinnamic acid amide 17 displayed 15-fold selectivity for M1 over M3 receptors. These findings will be useful in designing lead compounds for mAchRs and elucidating mechanisms of action of S. tangutica. PMID:28387362

  3. Muscarinic acetylcholine receptors: location of the ligand binding site

    SciTech Connect

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-05-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, /sup 3/H-propylbenzilycholine mustard aziridinium ion (/sup 3/H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that /sup 3/H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin.

  4. Biochemical and immunological studies of the Muscarinic acetylcholine receptor

    SciTech Connect

    Gainer, M.W.

    1985-01-01

    Muscarinic acetylcholine receptors were solubilized from bovine brain membranes with 3(3-cholamidopropyl)dimethylammonio)propanesulfonate (CHAPS). A combination of 10 mM CHAPS and 1 M NaCl solubilized 15-40% of the specific receptor binding sites from these membranes. The solubilized receptors displayed high affinity binding of the muscarinic antagonist, (/sup 3/H)quinuclidinyl benzilate with a K/sub D/ = 300 pM. In addition, the solubilized and retained guanyl nucleotide regulation of agonist binding characteristic of membrane bound receptors. Gel filtration experiments showed that solubilized receptors from cortex and cerebellum had different elution profiles. Analysis by sucrose density gradient centrifugation showed that receptors in the lower molecular weight peak sedimented with a coefficient of 5S. Receptors in the larger molecular weight peak sedimented to the bottom of the gradient. Attempts to purify receptors by chromatography on propylbenzilycholine Sepharose were unsuccessful. The technique used to attach the ligand to the solid support, however, was used to synthesize a PrBCM-BSA conjugate and the conjugate used as an antigen in the production of anti-ligand antibodies. Two anti-PrBCM monoclonal antibodies were isolated that recognize muscarinic but not nicotinic cholinergic ligands. The abilities of the antibodies to recognize other muscarinic ligands indicated the antibodies recognized a portion of PrBCM involved in binding to the receptor. Construction of an antibody affinity resin resulted in the purification of this fragment a minimum of 170 fold.

  5. Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

    SciTech Connect

    Large, T.H.; Cho, N.J.; De Mello, F.G.; Klein, W.L.

    1985-07-25

    Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.

  6. Nicotinic acetylcholine receptor agonist attenuates ILC2-dependent airway hyperreactivity

    PubMed Central

    Galle-Treger, Lauriane; Suzuki, Yuzo; Patel, Nisheel; Sankaranarayanan, Ishwarya; Aron, Jennifer L.; Maazi, Hadi; Chen, Lin; Akbari, Omid

    2016-01-01

    Allergic asthma is a complex and chronic inflammatory disorder that is associated with airway hyperreactivity (AHR) and driven by Th2 cytokine secretion. Type 2 innate lymphoid cells (ILC2s) produce large amounts of Th2 cytokines and contribute to the development of AHR. Here, we show that ILC2s express the α7-nicotinic acetylcholine receptor (α7nAChR), which is thought to have an anti-inflammatory role in several inflammatory diseases. We show that engagement of a specific agonist with α7nAChR on ILC2s reduces ILC2 effector function and represses ILC2-dependent AHR, while decreasing expression of ILC2 key transcription factor GATA-3 and critical inflammatory modulator NF-κB, and reducing phosphorylation of upstream kinase IKKα/β. Additionally, the specific α7nAChR agonist reduces cytokine production and AHR in a humanized ILC2 mouse model. Collectively, our data suggest that α7nAChR expressed by ILC2s is a potential therapeutic target for the treatment of ILC2-mediated asthma. PMID:27752043

  7. Nicotinic Acetylcholine Receptors at the Single-Channel Level.

    PubMed

    Bouzat, Cecilia; Sine, Steven M

    2017-03-05

    Over the past four decades, the patch clamp technique and nicotinic acetylcholine (nACh) receptors have established an enduring partnership. Like all good partnerships, each partner has proven significant in its own right, while their union has spurred innumerable advances in life science research. A member and prototype of the superfamily of pentameric ligand-gated ion channels, the nACh receptor is a chemo-electric transducer, binding nerve-released ACh and rapidly opening its channel to cation flow to elicit cellular excitation. A subject of a Nobel Prize in Physiology or Medicine, the patch clamp technique provides unprecedented resolution of currents through single ion channels in their native cellular environments. Here, focusing on muscle and α7 nACh receptors, we describe the extraordinary contribution of the patch clamp technique toward understanding how they activate in response to neurotransmitter, how subtle structural and mechanistic differences among nACh receptor subtypes translate into significant physiological differences, and how nACh receptors are being exploited as therapeutic drug targets.

  8. Acetylcholine-induced current in perfused rat myoballs

    PubMed Central

    1980-01-01

    Spherical "myoballs" were grown under tissue culture conditions from striated muscle of neonatal rat thighs. The myoballs were examined electrophysiologically with a suction pipette which was used to pass current and perfuse internally. A microelectrode was used to record membrane potential. Experiments were performed with approximately symmetrical (intracellular and extracellular) sodium aspartate solutions. The resting potential, acetylcholine (ACh) reversal potential, and sodium channel reversal potential were all approximately 0 mV. ACh-induced currents were examined by use of both voltage jumps and voltage ramps in the presence of iontophoretically applied agonist. The voltage-jump relaxations had a single exponential time-course. The time constant, tau, was exponentially related to membrane potential, increasing e-fold for 81 mV hyperpolarization. The equilibrium current- voltage relationship was also approximately exponential, from -120 to +81 mV, increasing e-fold for 104 mV hyperpolarization. The data are consistent with a first-order gating process in which the channel opening rate constant is slightly voltage dependent. The instantaneous current-voltage relationship was sublinear in the hyperpolarizing direction. Several models are discussed which can account for the nonlinearity. Evidence is presented that the "selectivity filter" for the ACh channel is located near the intracellular membrane surface. PMID:7381423

  9. Anesthetics Target Interfacial Transmembrane Sites in Nicotinic Acetylcholine Receptors

    PubMed Central

    Forman, Stuart A.; Chiara, David C.; Miller, Keith W.

    2014-01-01

    General anesthetics are a heterogeneous group of small amphiphilic ligands that interact weakly at multiple allosteric sites on many pentameric ligand gated ion channels (pLGICs), resulting in either inhibition, potentiation of channel activity, or both. Allosteric principles imply that modulator sites must change configuration and ligand affinity during receptor state transitions. Thus, general anesthetics and related compounds are useful both as state-dependent probes of receptor structure and as potentially selective modulators of pLGIC functions. This review focuses on general anesthetic sites in nicotinic acetylcholine receptors, which were among the first anesthetic-sensitive pLGIC experimental models studied, with particular focus on sites formed by transmembrane domain elements. Structural models place many of these sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (i.e. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface. PMID:25316107

  10. Acetylcholine-Atropine Interactions: Paradoxical Effects on Atrial Fibrillation Inducibility.

    PubMed

    Liu, Yu; Scherlag, Benjamin J; Fan, Youqi; Xia, Wenfang; Huang, He; Po, Sunny S

    2017-03-21

    Atropine (ATr) is well known as a cholinergic antagonist, however, at low concentrations ATr could paradoxically accentuate the parasympathetic actions of acetylcholine (ACh). In 22 pentobarbital anesthetized dogs, via a left and right thoracotomy, a leak proof barrier was attached to isolate the atrial appendages (AAs) from the rest of the atria. In Group 1(Ach+ATr+Ach), ACh, 100 mM, was placed on the AA followed by the application of ATr, 2mg/cc. The average AFdur was 17±7 minutes. After ATr was applied to the AA and ACh again tested, the AFdur was markedly attenuated (2±2 minutes, p<0.05). In Group 2 (ATr+Ach), ATr was initially applied to the AA followed by the application of ACh, 100 mM. There was no significant difference in AF duration (16±4 minutes vs 18±2 minutes, p=NS). The inhibitory effect of ATr on induced HR reduction (electrical stimulation of the anterior right ganglionated plexi and vagal nerves) was no difference between Groups 1 and 2. These observations suggest that when ATr is initially administered it attaches to the allosteric site of the muscarinic ACh receptor (M2 AChRs) leaving the orthosteric site free to be occupied by ACh. The M3 receptor that controls HR slowing does not show the same allosteric properties.

  11. Neuronal Nicotinic Acetylcholine Receptor Modulators Reduce Sugar Intake

    PubMed Central

    Shariff, Masroor; Quik, Maryka; Holgate, Joan; Morgan, Michael; Patkar, Omkar L.; Tam, Vincent; Belmer, Arnauld; Bartlett, Selena E.

    2016-01-01

    Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4β2 * and decreases α6β2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption. PMID:27028298

  12. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  13. Paroxysmal atrial fibrillation during intracoronary acetylcholine provocation test.

    PubMed

    Saito, Yuichi; Kitahara, Hideki; Shoji, Toshihiro; Tokimasa, Satoshi; Nakayama, Takashi; Sugimoto, Kazumasa; Fujimoto, Yoshihide; Kobayashi, Yoshio

    2016-12-22

    Intracoronary acetylcholine (ACh) provocation test is useful to diagnose vasospastic angina. However, paroxysmal atrial fibrillation (AF) often occurs during intracoronary ACh provocation test, leading to disabling symptoms. The aim of this study was to investigate the incidence and predictors of paroxysmal AF during the test. A total of 377 patients without persistent AF who underwent intracoronary ACh provocation test were included. Paroxysmal AF during ACh provocation test was defined as documented AF on electrocardiogram during the procedure. There were 31 patients (8%) with paroxysmal AF during the test. Of these, 11 (35%) required antiarrhythmic drugs, but none received electrical cardioversion. All of them recovered sinus rhythm within 48 h. At procedure, paroxysmal AF occurred mostly during provocation for the right coronary artery (RCA) rather than for the left coronary artery (LCA) (90 vs. 10%). Multivariate logistic regression analysis demonstrated that a history of paroxysmal AF (OR 4.38 CI 1.42-13.51, p = 0.01) and body mass index (OR 0.88 CI 0.78-0.99, p = 0.03) were independent predictors for occurrence of paroxysmal AF during intracoronary ACh provocation test. In conclusions, paroxysmal AF mostly occurs during ACh provocation test for the RCA, especially in patients with a history of paroxysmal AF and lower body mass index. It may be better to initially administer intracoronary ACh in the LCA when the provocation test is performed.

  14. In vitro modifications of rat NTE and other esterases by chemicals which induce delayed neurotoxicity in vivo.

    PubMed

    Blasco, R; Moreno, E; Sanz, P; Repetto, M

    1990-10-01

    A rat in vitro model has been developed which permits direct study of the biochemical mechanisms involved in delayed neurotoxicity induced by any chemical compound, not only organophosphates. Using rat brain homogenate, a parallel study on the activity of neurotoxic esterase (NTE) and total esterases (TE) compared the action of metamidophos, which is known to induce delayed neurotoxicity, and the synthetic fatty acid anilides, oleylanilide and linoleylanilide. Inhibition in the activity of NTE and TE, unrelated to the concentration and the incubation time assayed, was caused by metamidophos, while the anilides showed a 2-phase concentration-time dependent behaviour. This confirmed the results we previously obtained in vivo. In both cases the appearance of delayed neuropathy was related to modification of NTE activity. We concluded that phosphorylation of the enzyme may not be the only biochemical requirement for the development of delayed neurotoxicity syndromes in which modification of NTE is produced.

  15. Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus.

    PubMed

    Dong, Hao; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

    2017-02-28

    Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h.

  16. Extracellular and intracellular esterase processing of SCFA-hexosamine analogs: implications for metabolic glycoengineering and drug delivery.

    PubMed

    Mathew, Mohit P; Tan, Elaine; Shah, Shivam; Bhattacharya, Rahul; Adam Meledeo, M; Huang, Jun; Espinoza, Freddy A; Yarema, Kevin J

    2012-11-15

    This report provides a synopsis of the esterase processing of short chain fatty acid (SCFA)-derivatized hexosamine analogs used in metabolic glycoengineering by demonstrating that the extracellular hydrolysis of these compounds is comparatively slow (e.g., with a t(1/2) of ∼4 h to several days) in normal cell culture as well as in high serum concentrations intended to mimic in vivo conditions. Structure-activity relationship (SAR) analysis of common sugar analogs revealed that O-acetylated and N-azido ManNAc derivatives were more refractory against extracellular inactivation by FBS than their butanoylated counterparts consistent with in silico docking simulations of Ac(4)ManNAc and Bu(4)ManNAc to human carboxylesterase 1 (hCE1). By contrast, all analogs tested supported increased intracellular sialic acid production within 2h establishing that esterase processing once the analogs are taken up by cells is not rate limiting.

  17. Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

    PubMed Central

    Rajendran, Ranjith; Borghi, Elisa; Falleni, Monica; Perdoni, Federica; Tosi, Delfina; Lappin, David F.; O'Donnell, Lindsay; Greetham, Darren; Ramage, Gordon

    2015-01-01

    Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections. PMID:26092919

  18. The apeE Gene of Salmonella typhimurium Encodes an Outer Membrane Esterase Not Present in Escherichia coli

    PubMed Central

    Carinato, Maria E.; Collin-Osdoby, Patricia; Yang, Xioming; Knox, Tina M.; Conlin, Christopher A.; Miller, Charles G.

    1998-01-01

    Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine β-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674–680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli. PMID:9657991

  19. An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum

    NASA Astrophysics Data System (ADS)

    Colombres, Marcela; Garate, José A.; Lagos, Carlos F.; Araya-Secchi, Raúl; Norambuena, Patricia; Quiroz, Soledad; Larrondo, Luis; Pérez-Acle, Tomas; Eyzaguirre, Jaime

    2008-01-01

    The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 Å resolution (PDB 1G66). The enzyme possesses the α/β hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.

  20. Multiple resistance to pirimiphos-methyl and bifenthrin in Tribolium castaneum involves the activity of lipases, esterases, and laccase2.

    PubMed

    Julio, Alison Henrique Ferreira; Gigliolli, Adriana Aparecida Sinópolis; Cardoso, Kátia Aparecida Kern; Drosdoski, Sandro Daniel; Kulza, Rodrigo Amaral; Seixas, Flávio Augusto Vicente; Ruvolo-Takasusuki, Maria Claudia Colla; de Souza, Cristina Giatti Marques; Lapenta, Ana Silvia

    2017-05-01

    Several recent studies have elucidated the molecular mechanisms that confer insecticide resistance on insect pests. However, little is known about multiple resistance in red flour beetle (Tribolium castaneum) at molecular level. The multiple resistance is characterized as resistance to different classes of insecticides that have different target sites, and is mediated by several enzymatic systems. In this study, we investigated the biochemical and molecular mechanisms involved in multiple resistance of T. castaneum to bifenthrin (pyrethroid [Pyr]) and pirimiphos-methyl (organophosphate [Org]). We used artificial selection, biochemical and in silico approaches including structural computational biology. After five generations of artificial selection in the presence of bifenthrin (F5Pyr) or pirimiphos-methyl (F5Org), we found high levels of multiple resistance. The hierarchical enzymatic cluster revealed a pool of esterases (E), lipases (LIPs) and laccase2 (LAC2) potentially contributing to the resistance in different ways throughout development, after one or more generations in the presence of insecticides. The enzyme-insecticide interaction network indicated that E2, E3, LIP3, and LAC2 are enzymes potentially required for multiple resistance phenotype. Kinetic analysis of esterases from F5Pyr and F5Org showed that pirimiphos-methyl and specially bifenthrin promote enzyme inhibition, indicating that esterases mediate resistance by sequestering bifenthrin and pirimiphos-methyl. Our computational data were in accordance with kinetic results, indicating that bifenthrin has higher affinity at the active site of esterase than pirimiphos-methyl. We also report the capability of these insecticides to modify the development in T. castaneum. Our study provide insights into the biochemical mechanisms employed by T. castaneum to acquire multiple resistance.

  1. Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Starkov, Vladislav G; Osipov, Alexey V; Andreeva, Tatyana V; Filkin, Sergey Yu; Gorbacheva, Elena V; Astashev, Maxim E; Tsetlin, Victor I; Utkin, Yuri N

    2014-01-01

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

  2. Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

    PubMed

    Pereira, Mariana Rangel; Mercaldi, Gustavo Fernando; Maester, Thaís Carvalho; Balan, Andrea; Lemos, Eliana Gertrudes de Macedo

    2015-01-01

    Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

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

    PubMed

    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.

  4. Screening, purification, and characterization of a novel organic solvent-tolerant esterase, Lip2, from Monascus purpureus strain M7.

    PubMed

    Kang, Li-Jing; Meng, Zi-Tong; Hu, Chen; Zhang, Yan; Guo, Hai-Lun; Li, Qing; Li, Mu

    2017-03-01

    Organic solvent-tolerant esterases are proven to be excellent biocatalysts in chemical and pharmaceutical industries. A novel organic solvent-tolerant esterase gene, lip2, was isolated from filamentous fungi Monascus purpureus M7. The sequence analysis suggested that lip2 has a conserved "GDSL" motif near the active center. The multiple-sequence alignment and phylogenetic analysis revealed that Lip2 displayed two unique amino-acid sequence motifs that clearly separate it from any other previously described lipase family. After incubation in 20% methanol and ethanol for 3 h, the Lip2 displayed 190 and 180% residual activities, respectively. It retained 99-110% relative activity in 20% (v/v) hydrophilic organic solvents after incubation for 1 day. This esterase showed optimal activity at 40 °C and retained about 70% maximal activity at 60 °C. The enzyme also displayed more than 50% residual activity over a range of pH 5-11. In the presence of most of metal ions or additives, Lip2 retained most of the activity. These unique properties of Lip2 make it a promising as biocatalyst for industrial processes.

  5. Increased blood plasma hydrolysis of acetylsalicylic acid in type 2 diabetic patients: a role of plasma esterases.

    PubMed

    Gresner, Peter; Dolník, Martin; Waczulíková, Iveta; Bryszewska, Maria; Sikurová, Libusa; Watala, Cezary

    2006-02-01

    Hydrolysis of acetylsalicylic acid (ASA, aspirin), an antiplatelet drug commonly used in the prevention of stroke and myocardial infarction, seems to play a crucial role in its pharmacological action. Thirty-eight healthy volunteers and 38 type 2 diabetic patients were enrolled to test the hypothesis that the enhanced plasma degradation and lowered bioavailability of ASA in diabetic patients is associated with the attenuation of platelet response. Aspirin esterase activities were tested at pH 7.4 and 5.5. A significantly higher overall aspirin esterase activity was noted at pH 7.4 in the diabetic patients (P<0.003), corresponding to faster ASA hydrolysis (P<0.006). This increased activity was attributable to butyrylcholinesterase and probably to albumin, because it was effectively inhibited by eserine and 4-bis-nitrophenyl phosphate (P<0.01). No significant differences between control and diabetic subjects were found at pH 5.5 in either enzymatic activities or ASA hydrolysis rates. The enhanced plasma ASA degradation in diabetic subjects was significantly associated with the refractoriness of blood platelets to ASA (P<0.05) and modulated by plasma cholesterol (P<0.01). No direct effects of plasma pH or albumin were observed. In conclusion, higher aspirin esterase activity contributes to the lowered response of diabetic platelets to ASA-mediated antiplatelet therapy.

  6. Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation.

    PubMed

    Yabe, Ryotaro; Miura, Akane; Usui, Tatsuya; Mudrak, Ingrid; Ogris, Egon; Ohama, Takashi; Sato, Koichi

    2015-01-01

    Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.

  7. Effects of high hydrostatic pressure and temperature increase on Escherichia coli spp. and pectin methyl esterase inactivation in orange juice.

    PubMed

    Torres, E F; González-M, G; Klotz, B; Rodrigo, D

    2016-03-01

    The aim of this study was to evaluate the effect of high hydrostatic pressure treatment combined with moderate processing temperatures (25 ℃-50 ℃) on the inactivation of Escherichia coli O157: H7 (ATCC 700728), E. coli K12 (ATCC 23716), and pectin methyl esterase in orange juice, using pressures of 250 to 500 MPa with times ranging between 1 and 30 min. Loss of viability of E. coli O157:H7 increased significantly as pressure and treatment time increased, achieving a 6.5 log cycle reduction at 400 MPa for 3 min at 25 ℃ of treatment. With regard to the inactivation of pectin methyl esterase, the greatest reduction obtained was 90.05 ± 0.01% at 50 ℃ and 500 MPa of pressure for 15 min; therefore, the pectin methyl esterase enzyme was highly resistant to the treatments by high hydrostatic pressure. The results obtained in this study showed a synergistic effect between the high pressure and moderate temperatures in inactivating E. coli cells.

  8. Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation

    PubMed Central

    Pereira, Mariana Rangel; Mercaldi, Gustavo Fernando; Maester, Thaís Carvalho; Balan, Andrea; de Macedo Lemos, Eliana Gertrudes

    2015-01-01

    Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes. PMID:26214846

  9. Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris

    PubMed Central

    2012-01-01

    Background The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture. Results Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris, and the hydrolytic activity of the recombinant protein (OPE*) studied. After the initial screening of different clones expressing the enzyme, only one was selected for showing the highest production rate. Different culture conditions were tested to improve the expression of the recombinant enzyme. Complex media were better than minimal media for production, but in any case the levels of enzymatic activity were higher (7-fold in the best case) than those obtained from O. piceae. The purified enzyme had a molecular mass of 76 kDa, higher than that reported for the native enzyme under SDS-PAGE (60 kDa). Steady-state kinetic characterization of the recombinant protein showed improved catalytic efficiency for this enzyme as compared to the native one, for all the assayed substrates (p-nitrophenol, glycerol, and cholesterol esters). Different causes for this were studied, as the increased glycosylation degree of the recombinant enzyme, their secondary structures or the oxidation of methionine residues. However, none of these could explain the improvements found in the recombinant protein. N-terminal sequencing of OPE* showed that two populations of this enzyme were expressed, having either 6 or 8 amino acid residues more than the native one. This fact affected the aggregation behaviour of the recombinant protein, as was corroborated by analytical ultracentrifugation, thus improving the catalytic efficiency of this enzyme. Conclusion P. pastoris resulted to be an optimum biofactory for the

  10. Docking of 6-chloropyridazin-3-yl derivatives active on nicotinic acetylcholine receptors into molluscan acetylcholine binding protein (AChBP).

    PubMed

    Artali, Roberto; Bombieri, Gabriella; Meneghetti, Fiorella

    2005-04-01

    The crystal structure of Acetylcholine Binding Protein (AChBP), homolog of the ligand binding domain of nAChR, has been used as model for computational investigations on the ligand-receptor interactions of derivatives of 6-chloropyridazine substituted at C3 with 3,8-diazabicyclo[3.2.1]octane, 2,5-diazabicyclo[2.2.1]heptane and with piperazine and homopiperazine, substituted or not at N4. The ligand-receptor complexes have been analyzed by docking techniques using the binding site of HEPES complexed with AChBP as template. The good relationship between the observed binding affinity and the calculated docking energy confirms that this model provides a good starting point for understanding the binding domain of neuronal nicotinic receptors. An analysis of the possible factors significant for the ligand recognition has evidenced, besides the cation-pi interaction, the distance between the chlorine atom of the pyridazinyl group and the carbonylic oxygen of Leu B112 as an important parameter in the modulation of the binding energy.

  11. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

  12. The effect of inducers and inhibitors of urethane metabolism on its in vitro and in vivo metabolism in rats.

    PubMed

    Carlson, G P

    1994-12-09

    The activation of urethane (ethyl carbamate) is important in its exerting its carcinogenic effect. Rats were treated with inducers and inhibitors of urethane metabolism, and the conversion of [carbonyl-14C]urethane to 14CO2 in vivo was measured. The cytochrome P-450 inducers, phenobarbital and beta-naphthoflavone, and esterase inhibitor, paraoxon, were without effect while the CYP2E1 inhibitor, diethyldithiocarbamate, decreased metabolism to about 3% of control. Ethanol administered acutely inhibited urethane metabolism. Pyridine, shown previously to enhance this metabolism in microsomal preparations, greatly inhibited it in vivo. The discordant results between the in vitro and in vivo studies may be related to the presence of pyridine acting as an inhibitor in whole animals and suggest that caution is needed in extrapolating from in vitro results to in vivo implications.

  13. Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Alapafuja, Shakiru O.; Nikas, Spyros P.; Bharatan, Indu; Shukla, Vidyanand G.; Nasr, Mahmoud L.; Bowman, Anna L.; Zvonok, Nikolai; Li, Jing; Shi, Xiaomeng; Engen, John R.; Makriyannis, Alexandros

    2013-01-01

    Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs. PMID:23083016

  14. Circulating antibodies against nicotinic acetylcholine receptors in chagasic patients

    PubMed Central

    GOIN, J C; VENERA, G; BONINO, M BISCOGLIO DE JIMÉNEZ; STERIN-BORDA, L

    1997-01-01

    Human and experimental Chagas' disease causes peripheral nervous system damage involving neuromuscular transmission alterations at the neuromuscular junction. Additionally, autoantibodies directed to peripheral nerves and sarcolemmal proteins of skeletal muscle have been described. In this work, we analyse the ability of serum immunoglobulin factors associated with human chagasic infection to bind the affinity-purified nicotinic acetylcholine receptor (nAChR) from electric organs of Discopyge tschudii and to identify the receptor subunits involved in the interaction. The frequency of serum anti-nAChR reactivity assayed by dot-blot was higher in seropositive chagasic patients than in uninfected subjects. Purified IgG obtained from chagasic patients immunoprecipitated a significantly higher fraction of the solubilized nAChR than normal IgG. Furthermore, immunoblotting assays indicated that α and β are the main subunits involved in the interaction. Chagasic IgG was able to inhibit the binding of α-bungarotoxin to the receptor in a concentration-dependent manner, confirming the contribution of the α-subunit in the autoantibody-receptor interaction. The presence of anti-nAChR antibodies was detected in 73% of chagasic patients with impairment of neuromuscular transmission in conventional electromyographical studies, indicating a strong association between seropositive reactivity against nAChR and electromyographical abnormalities in chagasic patients. The chronic binding of these autoantibodies to the nAChR could induce a decrease in the population of functional nAChRs at the neuromuscular junction and consequently contribute to the electrophysiological neuromuscular alterations described in the course of chronic Chagas' disease. PMID:9367405

  15. Cell-surface translational dynamics of nicotinic acetylcholine receptors

    PubMed Central

    Barrantes, Francisco J.

    2014-01-01

    Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain. PMID

  16. Acetylcholine Receptor Organization in Membrane Domains in Muscle Cells

    PubMed Central

    Piguet, Joachim; Schreiter, Christoph; Segura, Jean-Manuel; Vogel, Horst; Hovius, Ruud

    2011-01-01

    Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction formation; however, it has not been demonstrated whether rapsyn is the only cause of receptor immobilization. Here, we used single-molecule tracking methods to investigate nAChR mobility in plasma membranes of myoblast cells during their differentiation to myotubes in the presence and absence of rapsyn. We found that in myoblasts the majority of nAChR were immobile and that ∼20% of the receptors showed restricted diffusion in small domains of ∼50 nm. In myoblasts devoid of rapsyn, the fraction of mobile nAChR was considerably increased, accompanied by a 3-fold decrease in the immobile population of nAChR with respect to rapsyn-expressing cells. Half of the mobile receptors were confined to domains of ∼120 nm. Measurements performed in heterologously transfected HEK cells confirmed the direct immobilization of nAChR by rapsyn. However, irrespective of the presence of rapsyn, about one-third of nAChR were confined in 300-nm domains. Our results show (i) that rapsyn efficiently immobilizes nAChR independently of other postsynaptic scaffold components; (ii) nAChR is constrained in confined membrane domains independently of rapsyn; and (iii) in the presence of rapsyn, the size of these domains is strongly reduced. PMID:20978122

  17. Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

    SciTech Connect

    Gracz, L.M.; Wang, W.; Parsons, S.M.

    1988-07-12

    Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP/sub 1/ synaptic vesicles banding at 1.055 g/mL actively transported (/sup 3/H)acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more (/sup 3/H)AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP/sub 1/ vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP/sub 2/ synaptic vesicle originally described in intact Torpedo marmorata electric organ. The properties of the receptor for vesamicol were studied by measuring binding of (/sup 3/H)vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP/sub 1/ vesicles the VP/sub 2/ vesicles had a ratio of (/sup 3/H)AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. The Hill coefficients ..cap alpha../sub H/ and equilibrium dissociation constants K for vesamicol binding to VP/sub 1/ and VP/sub 2/ vesicles were essentially the same. The positive Hill coefficient suggests that the vesamicol receptor exists as a homotropic oligomeric complex. The results demonstrate that VP/sub 1/ and VP/sub 2/ synaptic vesicles exhibit functional differences in the AcCh transport system, presumably as a result of regulatory phenomena.

  18. Neuronal nicotinic acetylcholine receptors are modulated by zinc.

    PubMed

    Vázquez-Gómez, Elizabeth; García-Colunga, Jesús

    2009-01-01

    It is known that zinc modulates nicotinic acetylcholine receptors (nAChRs). Here, we studied the effects of zinc on neuronal alpha4beta4 nAChRs, expressed in Xenopus oocytes and activated by nicotine. Membrane ion currents elicited by nicotine (10 nM to 100 microM) were enhanced by zinc (100 microM). Maximal zinc potentiation of the nicotine-activated current (2530%) occurred at 50 nM nicotine, and potentiation gradually decreased as the nicotine concentration increased. The EC(50) and IC(50) for the nicotine-activated current were 639 nM and 14.7 microM nicotine, respectively. Both parameters decreased in the presence of zinc to 160 nM and 4.6 microM, respectively, probably due to an increase of sensitivity of nAChRs for nicotine. We used different concentrations and durations of exposure to nicotine, due to desensitization of nAChRs directly depends on both these factors. With 500 nM nicotine and 20 min washing periods between nicotine applications, zinc potentiation remained constant, 901% for 2 min and 813% for 20 min of nicotine exposure. With continuous application of nicotine, zinc potentiation decreased as the time of nicotine exposure increased, 721% for 2 min and 254% for 48 min of nicotine exposure. Our results indicate that zinc-potentiating effects on alpha4beta4 nAChRs strongly depend on both concentration and time of exposure to nicotine, suggesting that zinc potentiation depends on the degree of desensitization.

  19. Generation of choline for acetylcholine synthesis by phospholipase D isoforms

    PubMed Central

    Zhao, Di; Frohman, Michael A; Blusztajn, Jan Krzysztof

    2001-01-01

    Dedication This article is dedicated to the memory of Sue Kim Hanson, a graduate student in the department of Pathology and Laboratory Medicine at Boston University School of Medicine, who perished in the terrorist attacks of September 11, 2001. Abstract Background In cholinergic neurons, the hydrolysis of phosphatidylcholine (PC) by a phospholipase D (PLD)-type enzyme generates some of the precursor choline used for the synthesis of the neurotransmitter acetylcholine (ACh). We sought to determine the molecular identity of the relevant PLD using murine basal forebrain cholinergic SN56 cells in which the expression and activity of the two PLD isoforms, PLD1 and PLD2, were experimentally modified. ACh levels were examined in cells incubated in a choline-free medium, to ensure that their ACh was synthesized entirely from intracellular choline. Results PLD2, but not PLD1, mRNA and protein were detected in these cells and endogenous PLD activity and ACh synthesis were stimulated by phorbol 12-myristate 13-acetate (PMA). Introduction of a PLD2 antisense oligonucleotide into the cells reduced PLD2 mRNA and protein expression by approximately 30%. The PLD2 antisense oligomer similarly reduced basal- and PMA-stimulated PLD activity and ACh levels. Overexpression of mouse PLD2 by transient transfection increased basal- (by 74%) and PMA-stimulated (by 3.2-fold) PLD activity. Moreover, PLD2 transfection increased ACh levels by 26% in the absence of PMA and by 2.1-fold in the presence of PMA. Overexpression of human PLD1 by transient transfection increased PLD activity by 4.6-fold and ACh synthesis by 2.3-fold in the presence of PMA as compared to controls. Conclusions These data identify PLD2 as the endogenous enzyme that hydrolyzes PC to generate choline for ACh synthesis in cholinergic cells, and indicate that in a model system choline generated by PLD1 may also be used for this purpose. PMID:11734063

  20. Developmental regulation of nicotinic acetylcholine receptors within midbrain dopamine neurons

    PubMed Central

    Azam, Layla; Chen, Yiling; Leslie, Frances M.

    2007-01-01

    We have combined anatomical and functional methodologies to provide a comprehensive analysis of the properties of nicotinic acetylcholine receptors (nAChRs) on developing dopamine (DA) neurons. Double-labeling in situ hybridization was used to examine the expression of nAChR subunit mRNAs within developing midbrain DA neurons. As brain maturation progressed there was a change in the pattern of subunit mRNA expression within DA neurons, such that α3 and α4 subunits declined and α6 mRNA increased. Although there were strong similarities in subunit mRNA expression in substantia nigra (SNc) and ventral tegmental area (VTA), there was higher expression of α4 mRNA in SNc than VTA at gestational day (G)15, and of α5, α6 and β3 mRNAs during postnatal development. Using a superfusion neurotransmitter release paradigm to functionally characterize nicotine-stimulated release of [3H]DA from striatal slices, the properties of the nAChRs on DA terminals were also found to change with age. Functional nAChRs were detected on striatal terminals at G18. There was a decrease in maximal release in the first postnatal week, followed by an increase in nicotine efficacy and potency during the second and third postnatal weeks. In the transition from adolescence (postnatal days (P) 30 and 40) to adulthood, there was a complex pattern of functional maturation of nAChRs in ventral, but not dorsal, striatum. In males, but not females, there were significant changes in both nicotine potency and efficacy during this developmental period. These findings suggest that nAChRs may play critical functional roles throughout DA neuronal maturation. PMID:17197101

  1. Nucleophilic and general acid catalysis at physiological pH by a designed miniature esterase.

    PubMed

    Nicoll, Andrew J; Allemann, Rudolf K

    2004-08-07

    A 31-residue peptide (Art-Est) was designed to catalyse the hydrolysis of p-nitrophenyl esters through histidine catalysis on the solvent exposed face of the alpha-helix of bovine pancreatic polypeptide. NMR spectroscopy indicated that Art-Est adopted a stable 3-dimensional structure in solution. Art-Est was an efficient catalyst with second order rate constants of up to 0.050 M(-1) s(-1). The activity of Art-Est was a consequence of the increased nucleophilicity of His-22, which had a reduced pK(a) value of 5.5 as a consequence of its interaction with His-18 and the positively charged Arg-25 and Arg-26. Mass spectrometry and NMR spectroscopy confirmed that the Art-Est catalysed hydrolysis of p-nitrophenyl esters proceeded through an acyl-enzyme intermediate. A solvent kinetic isotope effect of 1.8 indicated that the transition state preceding the acyl intermediate was stabilised through interaction with the protonated side-chain of His-18 and indicated a reaction mechanism similar to that generally observed for natural esterases. The involvement in the reaction of two histidine residues with different pK(a) values led to a bell-shaped dependence of the reaction rate on the pH of the solution. The catalytic behaviour of Art-Est indicated that designed miniature enzymes can act in a transparent mechanism based fashion with enzyme-like behaviour through the interplay of several amino acid residues.

  2. Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

    PubMed Central

    Grosse, Stephan; Bergeron, Hélène; Imura, Akihiro; Boyd, Jason; Wang, Shaozhao; Kubota, Kazuo; Miyadera, Akihiko; Sulea, Traian; Lau, Peter C. K.

    2010-01-01

    Summary There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)‐2‐benzyloxy‐propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50°C was outperformed by TtEST, a naturally occurring homologue with a Tm of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β‐fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis. PMID:21255307

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

    PubMed

    Hannam, Marie L; Hagger, Josephine A; Jones, Malcolm B; Galloway, Tamara S

    2008-03-01

    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 IC50 of 5 microg l(-1) for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 microg l(-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.

  4. Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl-saccharide esters.

    PubMed

    Kelle, Sebastian; Nieter, Annabel; Krings, Ulrich; Zelena, Katerina; Linke, Diana; Berger, Ralf G

    2016-11-01

    The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

  5. A Lactobacillus plantarum esterase active on a broad range of phenolic esters.

    PubMed

    Esteban-Torres, María; Landete, José María; Reverón, Inés; Santamaría, Laura; de las Rivas, Blanca; Muñoz, Rosario

    2015-05-01

    Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments.

  6. Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

    PubMed Central

    Xu, Lipeng; Liu, Zhongyuan; Xu, Kehui; Li, Shentao; Wen, Tingyi; Liu, Siguo; Pang, Hai

    2010-01-01

    Background It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined. Methodology/Principal Findings We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant β-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0–10.0 and at temperatures ≤40°C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C2–C8), and its suitable substrate was p-nitrophenyl caproate (C6) with optimal catalytic conditions of 39°C and pH 8.0. Conclusions/Significance Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis. PMID:20957207

  7. Identification and characterization of barley mutants lacking glycine decarboxylase and carboxyl esterase activities

    SciTech Connect

    Blackwell, R.; Lewis, K.; Lea, P. )

    1990-05-01

    A barley mutant has been isolated, from a selection of fifty air-sensitive seed-lines, using a standard gel stain technique which lacks carboxyl esterase activity, but has normal levels of carbonic anhydrase. In addition, two barley mutants lacking the ability to convert glycine to serine in the mitochondria, have been characterized. Both plants accumulate glycine in air and are unable to metabolize ({sup 14}C)glycine in the short-term. When ({sup 14}C)glycine was supplied over 2h LaPr 85/55 metabolized 90%, whereas the second mutant (LaPr 87/30) metabolized 10%. Results indicate that the mutation in LaPr 85/55 is almost certainly in the glycine transporter into the mitochondrion. The mutation in LaPr 87/30 has been shown, using western blotting, to be in both the P and H proteins, two of four proteins which comprise glycine decarboxylase (P, H, T and L).

  8. Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

    PubMed

    Petrovskaya, Lada E; Novototskaya-Vlasova, Ksenia A; Spirina, Elena V; Durdenko, Ekaterina V; Lomakina, Galina Yu; Zavialova, Maria G; Nikolaev, Evgeny N; Rivkina, Elizaveta M

    2016-05-01

    As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

  9. Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

    SciTech Connect

    Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.; Nichols, Joseph; Edwald, Elin; Chan, Jimmy; Ko, Mei-Chuan; Woods, James H.; Tesmer, John J.G.; Sunahara, Roger K.

    2012-03-15

    No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37 C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37 C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.

  10. Discovery a novel organic solvent tolerant esterase from Salinispora arenicola CNP193 through genome mining.

    PubMed

    Fang, Yaowei; Wang, Shujun; Liu, Shu; Jiao, Yuliang

    2015-09-01

    An esterase gene, encoding a 325-amino-acid protein (SAestA), was mined form obligate marine actinomycete strain Salinispora arenicola CNP193 genome sequence. Phylogenetic analysis of the deduced amino acid sequence showed that the enzyme belonged to the family IV of lipolytic enzymes. The gene was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The molecular weight of His-tagged SAestA is ∼38 kDa. SAestA-His6 was active in a temperature (5-40 °C) and pH range (7.0-11.0), and maximal activity was determined at pH 9.0 and 30 °C. The activity was severely inhibited by Hg(2+), Cu(2+), and Zn(2+). In particular, this enzyme showed remarkable stability in presence of organic solvents (25%, v/v) with log P>2.0 even after incubation for 7 days. All these characteristics suggested that SAestA may be a potential candidate for application in industrial processes in aqueous/organic media.

  11. Characterization of a novel enzyme-Starmerella bombicola lactone esterase (SBLE)-responsible for