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Sample records for acetylcholinesterase ache butyrylcholinesterase

  1. 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

  2. Dual inhibition of acetylcholinesterase and butyrylcholinesterase enzymes by allicin

    PubMed Central

    Kumar, Suresh

    2015-01-01

    Objectives: The brain of mammals contains two major form of cholinesterase enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The dual inhibition of these enzymes is considered as a promising strategy for the treatment of neurological disorder such as Alzheimer's disease (AD), senile dementia, ataxia, and myasthenia gravis. The present study was undertaken to explore the anticholinesterase inhibition property of allicin. Materials and Methods: An assessment of cholinesterase inhibition was carried out by Ellman's assay. Results: The present study demonstrates allicin, a major ingredient of crushed garlic (Allium sativum L.) inhibited both AChE and BuChE enzymes in a concentration-dependent manner. For allicin, the IC50 concentration was 0.01 mg/mL (61.62 μM) for AChE and 0.05 ± 0.018 mg/mL (308.12 μM) for BuChE enzymes. Conclusions: Allicin shows a potential to ameliorate the decline of cognitive function and memory loss associated with AD by inhibiting cholinesterase enzymes and upregulate the levels of acetylcholine (ACh) in the brain. It can be used as a new lead to target AChE and BuChE to upregulate the level of ACh which will be useful in alleviating the symptoms associated with AD. PMID:26288480

  3. Genetic Analysis of Collagen Q: Roles in Acetylcholinesterase and Butyrylcholinesterase Assembly and in Synaptic Structure and Function

    PubMed Central

    Feng, Guoping; Krejci, Eric; Molgo, Jordi; Cunningham, Jeanette M.; Massoulié, Jean; Sanes, Joshua R.

    1999-01-01

    Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ−/− mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ−/− muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ−/− neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity. PMID:10087275

  4. Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

    SciTech Connect

    Jbilo, O.; Barteles, C.F.; Chatonnet, A.; Toutant, J.P.; Lockridge, O.

    1994-12-31

    Tissue distribution of human acetyicholinesterase and butyryicholinesterase messenger RNA. 1 Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetyicholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

  5. Malathion, carbofuran and paraquat inhibit Bungarus sindanus (krait) venom acetylcholinesterase and human serum butyrylcholinesterase in vitro.

    PubMed

    Ahmed, Mushtaq; Rocha, João Batista T; Mazzanti, Cinthia M; Morsch, André L B; Cargnelutti, Denise; Corrêa, Maísa; Loro, Vânia; Morsch, Vera Maria; Schetinger, Maria R C

    2007-05-01

    Carbofuran and malathion, well known pesticides, and paraquat, a world widely used herbicide, were tested on acetylcholinesterase (AChE) from Bungarus sindanus venom and butyrylcholinesterase (BChE) from human serum. The calculated IC(50 )values for inhibition of venom enzyme by malathion, carbofuran and paraquat were 2.5, 0.14, and 0.16 microM, respectively. The values for inhibition of serum butyrylcholinesterase (BChE) were 3.5, 0.09 and 0.18 microM, respectively. Analysis of kinetic data indicated that the inhibition caused by malathion, carbofuran and paraquat was mixed for venom AChE. For BChE from human serum, the inhibition caused by malathion and paraquat was mixed and for carbofuran it was uncompetitive. The present results suggest a commercial paraquat preparation (a popular herbicide) inhibits cholinesterases with similar or higher potency than classical pesticide inhibitors. Furthermore, this inhibition was observed both in human serum and snake venom, a newly studied source of AChE.

  6. The interactions of azure B, a metabolite of methylene blue, with acetylcholinesterase and butyrylcholinesterase.

    PubMed

    Petzer, Anél; Harvey, Brian H; Petzer, Jacobus P

    2014-02-01

    Methylene blue (MB) is reported to possess diverse pharmacological actions and is attracting increasing attention for the treatment of neurodegenerative disorders such as Alzheimer's disease. Among the pharmacological actions of MB, is the significant inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These activities may, at least in part, underlie MB's beneficial effects in Alzheimer's disease. MB is metabolized to yield N-demethylated products of which azure B, the monodemethyl metabolite, is the predominant species. Azure B has been shown to be pharmacologically active and also possesses a variety of biological actions. Azure B therefore may contribute to the pharmacological profile of MB. Based on these considerations, the present study investigates the possibility that azure B may, similar to MB, act as an inhibitor of human AChE and BuChE. The results document that azure B inhibits AChE and BuChE with IC50 values of 0.486μM and 1.99μM, respectively. The results further show that azure B inhibits AChE and BuChE reversibly, and that the modes of inhibition are most likely competitive. Although the AChE and BuChE inhibitory activities of azure B are twofold and fivefold, respectively, less potent than those recorded for MB [IC50(AChE)=0.214μM; IC50(BuChE)=0.389μM] under identical conditions, azure B may be a contributor to MB's in vivo activation of the cholinergic system and beneficial effects in Alzheimer's disease.

  7. The interactions of azure B, a metabolite of methylene blue, with acetylcholinesterase and butyrylcholinesterase

    SciTech Connect

    Petzer, Anél; Harvey, Brian H.; Petzer, Jacobus P.

    2014-02-01

    Methylene blue (MB) is reported to possess diverse pharmacological actions and is attracting increasing attention for the treatment of neurodegenerative disorders such as Alzheimer's disease. Among the pharmacological actions of MB, is the significant inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These activities may, at least in part, underlie MB's beneficial effects in Alzheimer's disease. MB is metabolized to yield N-demethylated products of which azure B, the monodemethyl metabolite, is the predominant species. Azure B has been shown to be pharmacologically active and also possesses a variety of biological actions. Azure B therefore may contribute to the pharmacological profile of MB. Based on these considerations, the present study investigates the possibility that azure B may, similar to MB, act as an inhibitor of human AChE and BuChE. The results document that azure B inhibits AChE and BuChE with IC{sub 50} values of 0.486 μM and 1.99 μM, respectively. The results further show that azure B inhibits AChE and BuChE reversibly, and that the modes of inhibition are most likely competitive. Although the AChE and BuChE inhibitory activities of azure B are twofold and fivefold, respectively, less potent than those recorded for MB [IC{sub 50}(AChE) = 0.214 μM; IC{sub 50}(BuChE) = 0.389 μM] under identical conditions, azure B may be a contributor to MB's in vivo activation of the cholinergic system and beneficial effects in Alzheimer's disease. - Highlights: • Methylene blue (MB) is a known inhibitor of AChE and BuChE. • Azure B, the major metabolite of MB, also is an inhibitor of AChE and BuChE. • Azure B may be a contributor to MB's in vivo activation of the cholinergic system. • Azure B may contribute to MB's potential in Alzheimer's disease therapy.

  8. Calcium-activated butyrylcholinesterase in human skin protects acetylcholinesterase against suicide inhibition by neurotoxic organophosphates

    SciTech Connect

    Schallreuter, Karin U.; University of Bradford ). E-mail: K.Schallreuter@bradford.ac.uk; Gibbons, Nicholas C.J.; Elwary, Souna M.; Parkin, Susan M.; Wood, John M.

    2007-04-20

    The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5 x 10{sup -3}M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. {sup 45}Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H{sub 2}O{sub 2}-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8 m{sup 2} surface area with its calcium gradient in the 10{sup -3}M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue.

  9. Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of new tacrine-like analogues.

    PubMed

    Marco, J L; de los Ríos, C; Carreiras, M C; Baños, J E; Badía, A; Vivas, N M

    2001-03-01

    The synthesis and preliminary results for acetylcholinesterase and butyrylcholinesterase inhibition activity of a series of pyrano[2,3-b]quinolines (2, 3) and benzonaphthyridines (5, 6) derivatives are described. These molecules are tacrine-like analogues which have been prepared from readily available polyfunctionalized ethyl [6-amino-5-cyano-4H-pyrans and 6-amino-5-cyanopyridines]-3-carboxylates via Friedlander condensation with selected ketones. These compounds showed moderate acetylcholinesterase inhibition activity, the more potent (2e, 5b) being 6 times less active than tacrine. The butyrylcholinesterase activity of some of these molecules is also discussed.

  10. Rosmarinus officinalis L. leaf extract improves memory impairment and affects acetylcholinesterase and butyrylcholinesterase activities in rat brain.

    PubMed

    Ozarowski, Marcin; Mikolajczak, Przemyslaw L; Bogacz, Anna; Gryszczynska, Agnieszka; Kujawska, Malgorzata; Jodynis-Liebert, Jadwiga; Piasecka, Anna; Napieczynska, Hanna; Szulc, Michał; Kujawski, Radoslaw; Bartkowiak-Wieczorek, Joanna; Cichocka, Joanna; Bobkiewicz-Kozlowska, Teresa; Czerny, Boguslaw; Mrozikiewicz, Przemyslaw M

    2013-12-01

    Rosmarinus officinalis L. leaf as part of a diet and medication can be a valuable proposal for the prevention and treatment of dementia. The aim of the study was to assess the effects of subchronic (28-fold) administration of a plant extract (RE) (200 mg/kg, p.o.) on behavioral and cognitive responses of rats linked with acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity and their mRNA expression level in the hippocampus and frontal cortex. The passive avoidance test results showed that RE improved long-term memory in scopolamine-induced rats. The extract inhibited the AChE activity and showed a stimulatory effect on BuChE in both parts of rat brain. Moreover, RE produced a lower mRNA BuChE expression in the cortex and simultaneously an increase in the hippocampus. The study suggests that RE led to improved long-term memory in rats, which can be partially explained by its inhibition of AChE activity in rat brain.

  11. Postnatal growth hormone deficiency in growing rats causes marked decline in the activity of spinal cord acetylcholinesterase but not butyrylcholinesterase.

    PubMed

    Koohestani, Faezeh; Brown, Chester M; Meisami, Esmail

    2012-11-01

    The effects of growth hormone (GH) deficiency on the developmental changes in the abundance and activity of cholinesterase enzymes were studied in the developing spinal cord (SC) of postnatal rats by measuring the specific activity of acetylcholinesterase (AChE), a marker for cholinergic neurons and their synaptic compartments, and butyrylcholinesterase (BuChE), a marker for glial cells and neurovascular cells. Specific activities of these two enzymes were measured in SC tissue of 21- and 90 day-old (P21, weaning age; P90, young adulthood) GH deficient spontaneous dwarf (SpDwf) mutant rats which lack anterior pituitary and circulating plasma GH, and were compared with SC tissue of normal age-matched control animals. Assays were carried out for AChE and BuChE activity in the presence of their specific chemical inhibitors, BW284C51 and iso-OMPA, respectively. Results revealed that mean AChE activity was markedly and significantly reduced [28% at P21, 49% at P90, (p<0.01)] in the SC of GH deficient rats compared to age-matched controls. GH deficiency had a higher and more significant effect on AChE activity of the older (P90) rats than the younger ones (P21) ones. In contrast, BuChE activity in SC showed no significant changes in GH deficient rats at either of the two ages studied. Results imply that, in the absence of pituitary GH, the postnatal proliferation of cholinergic synapses in the rat SC, a CNS structure, where AChE activity is abundant, is markedly reduced during both the pre- and postweaning periods; more so in the postweaning than preweaning ages. In contrast, the absence of any effects on BuChE activity implies that GH does not affect the development of non-neuronal elements, e.g., glia, as much as the neuronal and synaptic compartments of the developing rat SC.

  12. Local salt substitutes “Obu-otoyo” activate acetylcholinesterase and butyrylcholinesterase and induce lipid peroxidation in rat brain

    PubMed Central

    Oboh, Ganiyu; Ademiluyi, Adedayo O.

    2015-01-01

    Evidence has shown that ingestion of heavy metals can lead to neurodegenerative diseases. This study aimed to investigate the neurotoxic potential of salt substitutes (Obu-Otoyo); salt A (made by burning palm kernel shaft then soaked in water overnight and the extract from the resulting residue is used as the salt substitute) and salt B (an unrefined salt mined from a local site at Ilobu town, Osun-State, Nigeria) by assessing their effect on some key enzymes linked with neurodegenerative disease [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities] as well as on malondialdehyde (MDA) content of the rat brain. Salt substitutes were fed to normal rats as dietary inclusion at doses of 0.5 and 1.0% for 30 days. Thereafter, the effect of the salt substitutes on AChE and BChE activities as well as on MDA level in the rat brain was determined. The results revealed that the salt substitutes caused a significant (p<0.05) increase in both AChE and BChE activity and also induced lipid peroxidation in the brain of rats in vivo as well as under in vitro condition in a dose-dependent manner. The effect of the salt substitutes on AChE and BChE activities could be attributed to the presence of some toxic heavy metals. Therefore, the ability of the salt substitutes to induce lipid peroxidation and activate AChE and BChE activities could provide some possible mechanism for their neurotoxic effect. PMID:27486373

  13. Local salt substitutes "Obu-otoyo" activate acetylcholinesterase and butyrylcholinesterase and induce lipid peroxidation in rat brain.

    PubMed

    Akinyemi, Ayodele J; Oboh, Ganiyu; Ademiluyi, Adedayo O

    2015-09-01

    Evidence has shown that ingestion of heavy metals can lead to neurodegenerative diseases. This study aimed to investigate the neurotoxic potential of salt substitutes (Obu-Otoyo); salt A (made by burning palm kernel shaft then soaked in water overnight and the extract from the resulting residue is used as the salt substitute) and salt B (an unrefined salt mined from a local site at Ilobu town, Osun-State, Nigeria) by assessing their effect on some key enzymes linked with neurodegenerative disease [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities] as well as on malondialdehyde (MDA) content of the rat brain. Salt substitutes were fed to normal rats as dietary inclusion at doses of 0.5 and 1.0% for 30 days. Thereafter, the effect of the salt substitutes on AChE and BChE activities as well as on MDA level in the rat brain was determined. The results revealed that the salt substitutes caused a significant (p<0.05) increase in both AChE and BChE activity and also induced lipid peroxidation in the brain of rats in vivo as well as under in vitro condition in a dose-dependent manner. The effect of the salt substitutes on AChE and BChE activities could be attributed to the presence of some toxic heavy metals. Therefore, the ability of the salt substitutes to induce lipid peroxidation and activate AChE and BChE activities could provide some possible mechanism for their neurotoxic effect.

  14. An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon

    SciTech Connect

    Shenouda, Josephine; Green, Paula; Sultatos, Lester

    2009-12-01

    Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of alpha/beta-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005-3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis-Menten kinetics, with a K{sub m} of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis-Menten kinetics, with a k{sub i} of 3048 nM{sup -1} h{sup -1}, and a K{sub D} of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k{sub i} increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k{sub i}s of 1.2 and 19.3 nM{sup -1} h{sup -1}, respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

  15. Circannual rhythms of acetylcholinesterase (AChE) activity in the freshwater fish Cnesterodon decemmaculatus.

    PubMed

    Menéndez-Helman, Renata J; Ferreyroa, Gisele V; dos Santos Afonso, Maria; Salibián, Alfredo

    2015-01-01

    The use of biomarkers as a tool to assess responses of organisms exposed to pollutants in toxicity bioassays, as well as in aquatic environmental risk assessment protocols, requires the understanding of the natural fluctuation of the particular biomarker. The aim of this study was to characterize the intrinsic variations of acetylcholinesterase (AChE) activity in tissues of a native freshwater teleost fish to be used as biomarker in toxicity tests, taking into account both seasonal influence and fish size. Specific AChE activity was measured by the method of Ellman et al. (1961) in homogenates of fish anterior section finding a seasonal variability. The highest activity was observed in summer, decreasing significantly below 40% in winter. The annual AChE activity cycle in the anterior section was fitted to a sinusoidal function with a period of 11.2 months. Moreover, an inverse relationship between enzymatic activity and the animal size was established. The results showed that both the fish length and seasonal variability affect AChE activity. AChE activity in fish posterior section showed a similar trend to that in the anterior section, while seasonal variations of the activity in midsection were observed but differences were not statistically significant. In addition, no relationship between AChE and total tissue protein was established in the anterior and posterior sections suggesting that the circannual rhythms observed are AChE-specific responses. Results highlight the importance of considering both the fish size and season variations to reach valid conclusions when AChE activity is employed as neurotoxicity biomarker.

  16. Synthesis, biological evaluation and molecular modelling of diversely functionalized heterocyclic derivatives as inhibitors of acetylcholinesterase/butyrylcholinesterase and modulators of Ca2+ channels and nicotinic receptors.

    PubMed

    Marco, José L; de los Ríos, Cristóbal; García, Antonio G; Villarroya, Mercedes; Carreiras, M Carmo; Martins, Carla; Eleutério, Ana; Morreale, Antonio; Orozco, M; Luque, F Javier

    2004-05-01

    The synthesis and the biological activity of compounds 5-40 as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca(2+) channels and nicotinic receptors, are described. These molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedländer reaction with selected cycloalkanones. These compounds are moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the 'oxazolo-tacrine' derivatives are devoid of any activity. All compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca(2+) channels, and three of them, 64, 19 and 67, the non-L type of Ca(2+) channels. Molecular modelling studies suggest that these compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both, the catalytic and peripheral binding sites of the enzyme.

  17. Biological evaluation of synthetic α,β-unsaturated carbonyl based cyclohexanone derivatives as neuroprotective novel inhibitors of acetylcholinesterase, butyrylcholinesterase and amyloid-β aggregation.

    PubMed

    Zha, Gao-Feng; Zhang, Cheng-Pan; Qin, Hua-Li; Jantan, Ibrahim; Sher, Muhammad; Amjad, Muhammad Wahab; Hussain, Muhammad Ajaz; Hussain, Zahid; Bukhari, Syed Nasir Abbas

    2016-05-15

    A series of new α,β-unsaturated carbonyl-based cyclohexanone derivatives was synthesized by simple condensation method and all compounds were characterized by using various spectroscopic techniques. New compounds were evaluated for their effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds were also screened for in vitro cytotoxicity and for inhibitory activity for self-induced Aβ1-42 aggregation. The effect of these compounds against amyloid β-induced cytotoxicity was also investigated. The findings of in vitro experiment revealed that most of these compounds exhibited potent inhibitory activity against AChE and self-induced Aβ1-42 aggregation. The compound 3o exhibited best AChE (IC50=0.037μM) inhibitory potential. Furthermore, compound 3o disassembled the Aβ fibrils produced by self-induced Aβ aggregation by 76.6%. Compounds containing N-methyl-4-piperidone linker, showed high acetylcholinesterase and self-induced Aβ aggregation inhibitory activities as compared to reference drug donepezil. The pre-treatment of cells with synthetic compounds protected them against Aβ-induced cell death by up to 92%. Collectively, these findings suggest that some compounds from this series have potential to be promising multifunctional agents for AD treatment and our study suggest the cyclohexanone derivatives as promising new inhibitors for AChE and BuChE, potentially useful to treat neurodegenerative diseases.

  18. Chemical synthesis of two series of nerve agent model compounds and their stereoselective interaction with human acetylcholinesterase and human butyrylcholinesterase.

    PubMed

    Barakat, Nora H; Zheng, Xueying; Gilley, Cynthia B; MacDonald, Mary; Okolotowicz, Karl; Cashman, John R; Vyas, Shubham; Beck, Jeremy M; Hadad, Christopher M; Zhang, Jun

    2009-10-01

    Both G and V type nerve agents possess a center of chirality about phosphorus. The S(p) enantiomers are generally more potent inhibitors than their R(p) counterparts toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). To develop model compounds with defined centers of chirality that mimic the target nerve agent structures, we synthesized both the S(p) and the R(p) stereoisomers of two series of G type nerve agent model compounds in enantiomerically enriched form. The two series of model compounds contained identical substituents on the phosphorus as the G type agents, except that thiomethyl (CH(3)-S-) and thiocholine [(CH(3))(3)NCH(2)CH(2)-S-] groups were used to replace the traditional nerve agent leaving groups (i.e., fluoro for GB, GF, and GD and cyano for GA). Inhibition kinetic studies of the thiomethyl- and thiocholine-substituted series of nerve agent model compounds revealed that the S(p) enantiomers of both series of compounds showed greater inhibition potency toward AChE and BChE. The level of stereoselectivity, as indicated by the ratio of the bimolecular inhibition rate constants between S(p) and R(p) enantiomers, was greatest for the GF model compounds in both series. The thiocholine analogues were much more potent than the corresponding thiomethyl analogues. With the exception of the GA model compounds, both series showed greater potency against AChE than BChE. The stereoselectivity (i.e., S(p) > R(p)), enzyme selectivity, and dynamic range of inhibition potency contributed from these two series of compounds suggest that the combined application of these model compounds will provide useful research tools for understanding interactions of nerve agents with cholinesterase and other enzymes involved in nerve agent and organophosphate pharmacology. The potential of and limitations for using these model compounds in the development of biological therapeutics against nerve agent toxicity are also discussed.

  19. Synthesis and in vitro acetylcholinesterase and butyrylcholinesterase inhibitory potential of hydrazide based Schiff bases.

    PubMed

    Rahim, Fazal; Ullah, Hayat; Taha, Muhammad; Wadood, Abdul; Javed, Muhammad Tariq; Rehman, Wajid; Nawaz, Mohsan; Ashraf, Muhammad; Ali, Muhammad; Sajid, Muhammad; Ali, Farman; Khan, Muhammad Naseem; Khan, Khalid Mohammed

    2016-10-01

    To discover multifunctional agents for the treatment of Alzheimer's disease, a series of hydrazide based Schiff bases were designed and synthesized based on multitarget-directed strategy. We have synthesized twenty-eight analogs of hydrazide based Schiff bases, characterized by various spectroscopic techniques and evaluated in vitro for acetylcholinesterase and butyrylcholinesterase inhibition. All compounds showed varied degree of acetylcholinesterase and butyrylcholinesterase inhibition when compared with standard Eserine. Among the series, compounds 10, 3 and 24 having IC50 values 4.12±0.01, 8.12±0.01 and 8.41±0.06μM respectively showed potent acetylcholinesterase inhibition when compared with Eserine (IC50=0.85±0.0001μM). Three compounds 13, 24 and 3 having IC50 values 6.51±0.01, 9.22±0.07 and 37.82±0.14μM respectively showed potent butyrylcholinesterase inhibition by comparing with eserine (IC50=0.04±0.0001μM). The remaining compounds also exhibited moderate to weak inhibitory potential. Structure activity relationship has been established. Through molecular docking studies the binding interaction was confirmed.

  20. Naturally Occurring Genetic Variants of Human Acetylcholinesterase and Butyrylcholinesterase and Their Potential Impact on the Risk of Toxicity from Cholinesterase Inhibitors

    PubMed Central

    2016-01-01

    Acetylcholinesterase (AChE) is the physiologically important target for organophosphorus toxicants (OP) including nerve agents and pesticides. Butyrylcholinesterase (BChE) in blood serves as a bioscavenger that protects AChE in nerve synapses from inhibition by OP. Mass spectrometry methods can detect exposure to OP by measuring adducts on the active site serine of plasma BChE. Genetic variants of human AChE and BChE do exist, but loss of function mutations have been identified only in the BCHE gene. The most common AChE variant, His353Asn (H322N), also known as the Yt blood group antigen, has normal AChE activity. The most common BChE variant, Ala567Thr (A539T) or the K-variant in honor of Werner Kalow, has 33% reduced plasma BChE activity. The genetic variant most frequently associated with prolonged response to muscle relaxants, Asp98Gly (D70G) or atypical BChE, has reduced activity and reduced enzyme concentration. Early studies in young, healthy males, performed at a time when it was legal to test nerve agents in humans, showed that individuals responded differently to the same low dose of sarin with toxic symptoms ranging in severity from minimal to moderate. Additionally, animal studies indicated that BChE protects from toxicants that have a higher reactivity with AChE than with BChE (e.g., nerve agents) but not from toxicants that have a higher reactivity with BChE than with AChE (e.g., OP pesticides). As a corollary, we hypothesize that individuals with genetic variants of BChE may be at increased risk of toxicity from nerve agents but not from OP pesticides. PMID:27551784

  1. The acetylcholinesterase (AChE) inhibition analysis of medaka (Oryzias latipes) in the exposure of three insecticides.

    PubMed

    Zhu, Jianping; Huan, Cheng; Si, Guiyun; Yang, Haitang; Yin, Li; Ren, Qing; Ren, Baixiang; Fu, Rongshu; Miao, Mingsheng; Ren, Zongming

    2015-03-01

    The continuous effects on Acetylcholinesterase (AChE) activity of medaka (Oryzias latipes) caused by dichlorvos, methomyl and deltamethrin in vivo were investigated, and the trends of AChE activity inhibition due to the influence of these insecticides were discussed. The LC50-24h of dichlorvos, methomyl and deltamethrin on medaka were 2.3 mg/L, 0.2 mg/L, and 2.9×10(-3) mg/L respectively. The result suggested that at the beginning of the exposure, the AChE activity might increase, and the AChE activity in dead individuals was obviously lower than the live individuals. Though the de novo synthesis of AChE in medaka might help the AChE activity recover, the trends during the exposure in different treatments were downward, and it showed both exposure time and concentration dependent. Meanwhile, higher temperature might cause the AChE inhibition earlier due to the higher metabolic rate. Therefore, as a specific biomarker for organophosphate, carbamate pesticides and pyrethroids, the degree of the AChE inhibition with in vivo conditions is a good tool in continuous monitoring of insecticides, which may induce the nerve conduction disorders.

  2. A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method.

    PubMed

    Demirci, Gökhan; Doğaç, Yasemin İspirli; Teke, Mustafa

    2015-11-01

    In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme-specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25-65 °C), optimum pH (3-10), thermal stability (4-70 °C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE.

  3. Effect of Acetylcholinesterase and Butyrylcholinesterase on Intrauterine Insemination, Contribution to Inflammations, Oxidative Stress and Antioxidant Status; A Preliminary Report

    PubMed Central

    Haghnazari, Lida; Vaisi-Raygani, Asad; Keshvarzi, Farahnaz; Ferdowsi, Farivar; Goodarzi, Massoud; Rahimi, Zohreh; Baniamerian, Hossin; Tavilani, Haidar; Vaisi-Raygani, Hadis; Vaisi-Raygani, Hessam; Pourmotabbed, Tayehbeh

    2016-01-01

    Background: Oxidative stress affects women fertility and influences on the sperm quality by alterating activities of cholinesterases, a molecular marker of stress-related infertility. The aim of the present study was to investigate the role of acetyl-cholinesterase (AChE), butyrylcholinesterase (BuChE) activities and phenotypes in patients with unexplained infertility (idiopathic). It’s possible association with inflammation marker C-reactive protein (CRP) and other oxidative stress markers, i.e. before and after intra uterine insemination (IUI). Methods: In this study, blood samples of 60 patients with unexplained infertility were collected the day before and 24 hr after IUI (between 8 AM and 9 AM after the overnight fasting) and activities of BuChE, AChE, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GpX) and serum levels of thiol proteins (TP), C-reactive protein (CRP), total antioxidant capacity (TAC) were measured. Statistical significance was assumed at p<0.05. Results: Before IUI, there was a significant (p=0.048) positive correlation between BuChE activity and plasma TAC and a significant difference in the CAT activity between various BuChE (UU and non-UU) phenotypes. However, after IUI, a significant negative correlation between the AChE activity and BuChE activity was found (p=0.045) and the level of RBC AChE activity was significantly reduced (382.4± 163.19 vs. 586.7±384 IU/grHb, p=0.025). Meanwhile, after IUI, the activities of SOD (1568±847.5 IU/grHb vs. 1126±229.3, p=0.031) and CAT (310±53.4 IU/grHb vs. 338±73, p=0.025) were increased. Conclusion: This study suggests that decline in cholinesterases activities may be responsible for stimulation of oxidative stress and inflammation and reduction in fertility rates by IUI. PMID:27478769

  4. In Vitro and In Vivo Metabolism and Inhibitory Activities of Vasicine, a Potent Acetylcholinesterase and Butyrylcholinesterase Inhibitor

    PubMed Central

    Liu, Wei; Shi, Xiaoyuan; Yang, Yadi; Cheng, Xuemei; Liu, Qing; Han, Han; Yang, Baohua; He, Chunyong; Wang, Yongli; Jiang, Bo; Wang, Zhengtao; Wang, Changhong

    2015-01-01

    Vasicine (VAS), a potential natural cholinesterase inhibitor, exhibited promising anticholinesterase activity in preclinical models and has been in development for treatment of Alzheimer’s disease. This study systematically investigated the in vitro and in vivo metabolism of VAS in rat using ultra performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry. A total of 72 metabolites were found based on a detailed analysis of their 1H- NMR and 13C NMR data. Six key metabolites were isolated from rat urine and elucidated as vasicinone, vasicinol, vasicinolone, 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-yl hydrogen sulfate, 9-oxo-1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-yl hydrogen sulfate, and 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-β-D-glucuronide. The metabolic pathway of VAS in vivo and in vitro mainly involved monohydroxylation, dihydroxylation, trihydroxylation, oxidation, desaturation, sulfation, and glucuronidation. The main metabolic soft spots in the chemical structure of VAS were the 3-hydroxyl group and the C-9 site. All 72 metabolites were found in the urine sample, and 15, 25, 45, 18, and 11 metabolites were identified from rat feces, plasma, bile, rat liver microsomes, and rat primary hepatocyte incubations, respectively. Results indicated that renal clearance was the major excretion pathway of VAS. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of VAS and its main metabolites were also evaluated. The results indicated that although most metabolites maintained potential inhibitory activity against AChE and BChE, but weaker than that of VAS. VAS undergoes metabolic inactivation process in vivo in respect to cholinesterase inhibitory activity. PMID:25849329

  5. Prediction of comparative inhibition efficiency for a novel natural ligand, galangin against human brain acetylcholinesterase, butyrylcholinesterase and 5-lipoxygenase: a neuroinformatics study.

    PubMed

    Shaikh, Sibhghatulla; Ahmad, Syed S; Ansari, Mohammad A; Shakil, Shazi; Rizvi, Syed M D; Shakil, Shahnawaz; Tabrez, Shams; Akhtar, Salman; Kamal, Mohammad A

    2014-04-01

    The present study elucidates molecular interactions of human acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and 5-lipoxygenase (5-LPO) with a novel natural ligand Galangin (GAL); and also with the well-known ligands Bisnorcymserine (BNC) and Cymserine for comparison. Docking between these ligands and enzymes were performed using 'Autodock4.2'. It was found that hydrophobic interactions play an important role in the correct positioning of BNC within the 'catalytic site' of AChE, BuChE and 5-LPO to permit docking while hydrogen bonds are significant in case of cymserine for the same. However, only polar interactions are significant in the correct positioning of GAL within the 'catalytic site' of AChE, BuChE and 5-LPO to permit docking. Such information may aid in the design of versatile AChE, BuChE and 5 LPO-inhibitors, and is expected to aid in safe clinical use of above ligands. Scope still remains in the determination of the three-dimensional structure of AChE-GAL, BuChE-GAL and 5-LPO-GAL complex by X-ray crystallography to certify the described data. Moreover, the present study confirms that GAL is a more efficient inhibitor of human brain AChE compared to BNC and cymserine, while in case of 5-LPO and human brain BuChE, BNC is a more efficient inhibitor compared to GAL and cymserine with reference to ΔG and Ki values.

  6. Novel assay utilizing fluorochrome-tagged physostigmine (Ph-F) to in situ detect active acetylcholinesterase (AChE) induced during apoptosis.

    PubMed

    Huang, Xuan; Lee, Brian; Johnson, Gary; Naleway, John; Guzikowski, Anthony; Dai, Wei; Darzynkiewicz, Zbigniew

    2005-01-01

    It was recently reported that acetylcholinesterase (AChE) is expressed in cells undergoing apoptosis and that its presence is essential for assembly of the apoptosome and subsequent caspase-9 activation. To obtain a marker of active AChE that could assay this enzyme in live intact cells and be applicable to fluorescence microscopy and cytometry, the fluorescein-tagged physostigmine (Ph-F), high affinity ligand (inhibitor) reactive with the active center of AChE, was constructed and tested for its ability to in situ label AChE and measure its induction during apoptosis. Ph-F inhibited cholinesterase activity in vitro (IC50 = 10(-6) and 5 x 10(-6) M for equine butyrylcholinesterase and human erythrocyte AChE, respectively) and was a selective marker of cells and structures that were AChE-positive. Thus, exposure of mouse bone marrow cells to Ph-F resulted in the exclusive labeling of megakaryocytes, and of the diaphragm muscle, preferential labeling of the nerve-muscle junctions (end-plates). During apoptosis of carcinoma HeLa cells and leukemic HL-60 or Jurkat cells triggered either by the DNA topoisomerase 1 inhibitor topotecan (TPT) or by oxidative stress (H2O2), the cells become reactive with Ph-F. Their Ph-F derived fluorescence was measured by flow and laser scanning cytometry. The appearance of Ph-F binding sites during apoptosis was preceded by the loss of mitochondrial potential, was concurrent with the presence of activated caspases, and was followed by loss of membrane integrity. At a very early stage of apoptosis, when nucleolar segregation was apparent, the Ph-F binding sites were distinctly localized within the nucleolus and at later stages of apoptosis in the cytoplasm. During apoptosis triggered by TPT, Ph-F binding was preferentially induced in S-phase cells. Our data on megakaryocytes and end-plates indicate that Ph-F reacts with active sites of AChE, and can be used to reveal the presence of this enzyme in live cells and possibly to study its

  7. Effect of pharmaceuticals exposure on acetylcholinesterase (AchE) activity and on the expression of AchE gene in the monogonont rotifer, Brachionus koreanus.

    PubMed

    Rhee, Jae-Sung; Kim, Bo-Mi; Jeong, Chang-Bum; Park, Heum Gi; Leung, Kenneth Mei Yee; Lee, Young-Mi; Lee, Jae-Seong

    2013-11-01

    Pharmaceuticals are widely used in human and veterinary medicine. However, they are emerging as a significant contaminant in aquatic environments through wastewater. Due to the persistent and accumulated properties of pharmaceuticals via the food web, their potential harmful effects on aquatic animals are a great concern. In this study, we investigated the effects of six pharmaceuticals: acetaminophen, ATP; atenolol, ATN; carbamazepine, CBZ; oxytetracycline, OTC; sulfamethoxazole, SMX; and trimethoprim, TMP on acetylcholinesterase (AChE; EC 3.1.1.7) activity and its transcript expression with chlorpyrifos (as a positive control) in the monogonont rotifer, Brachionus koreanus. ATP, CBZ, and TMP exposure also remarkably inhibited Bk-AChE activity at 100 μg/L (24 h) and 1000 μg/L (12 h and 24 h). ATP, CBZ, and TMP exposure showed a significant decrease in the Bk-AChE mRNA level in a concentration-dependent manner. However, in the case of OTC and SMX, a slight decrease in Bk-AChE mRNA expression was found but only at the highest concentration. The time-course experiments showed that ATP positively induced Bk-AChE mRNA 12 h after exposure at both 100 and 1000 μg/L, while the Bk-AChE mRNA expression was significantly downregulated over 6 to 24 h after exposure to 1000 μg/L of CBZ, OTC, SMX, and TMP. Our findings suggest that Bk-AChE would be a useful biomarker for risk assessment of pharmaceutical compounds as an early signal of their toxicity in aquatic environments. Particularly, ATP, CBZ, and TMP may have a toxic cholinergic effect on rotifer B. koreanus by inhibiting AChE activity.

  8. Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion.

    PubMed

    Camp, Shelley; Zhang, Limin; Marquez, Michael; de la Torre, Brian; Long, Jeffery M; Bucht, Goran; Taylor, Palmer

    2005-12-15

    AChE is an alternatively spliced gene. Exons 2, 3 and 4 are invariantly spliced, and this sequence is responsible for catalytic function. The 3' alternatively spliced exons, 5 and 6, are responsible for AChE disposition in tissue [J. Massoulie, The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11 (3) (2002) 130-143; Y. Li, S. Camp, P. Taylor, Tissue-specific expression and alternative mRNA processing of the mammalian acetylcholinesterase gene. J. Biol. Chem. 268 (8) (1993) 5790-5797]. The splice to exon 5 produces the GPI anchored form of AChE found in the hematopoietic system, whereas the splice to exon 6 produces a sequence that binds to the structural subunits PRiMA and ColQ, producing AChE expression in brain and muscle. A third alternative RNA species is present that is not spliced at the 3' end; the intron 3' of exon 4 is used as coding sequence and produces the read-through, unanchored form of AChE. In order to further understand the role of alternative splicing in the expression of the AChE gene, we have used homologous recombination in stem cells to produce gene specific deletions in mice. Alternatively and together exon 5 and exon 6 were deleted. A cassette containing the neomycin gene flanked by loxP sites was used to replace the exon(s) of interest. Tissue analysis of mice with exon 5 deleted and the neomycin cassette retained showed very low levels of AChE expression, far less than would have been anticipated. Only the read-through species of the enzyme was produced; clearly the inclusion of the selection cassette disrupted splicing of exon 4 to exon 6. The selection cassette was then deleted in exon 5, exon 6 and exons 5 + 6 deleted mice by breeding to Ella-cre transgenic mice. AChE expression in serum, brain and muscle has been analyzed. Another AChE gene targeted mouse strain involving a region in the first intron, found to be critical for AChE expression in muscle cells [S. Camp, L. Zhang, M. Marquez, B

  9. Acetylcholinesterases of Rhipicephalus (Boophilus) microplus – Multiple gene expression presents an opportune model system for elucidation of multiple functions of AChEs.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acetylcholinesterase (AChE) is a key neural enzyme of both vertebrates and invertebrates, and is the biochemical target of organophosphate and carbamate pesticides for invertebrates, as well as vertebrate nerve agents, e.g., soman, tabun, VX, and others. AChE inhibitors are also key drugs among thos...

  10. Alkaloids from Peumus boldus and their acetylcholinesterase, butyrylcholinesterase and prolyl oligopeptidase inhibition activity.

    PubMed

    Hošt'álková, Anna; Opletal, Lubomír; Kuneš, Jiří; Novák, Zdeněk; Hrabinová, Martina; Chlebek, Jakub; Čegan, Lukáš; Cahlíková, Lucie

    2015-04-01

    Eleven isoquinoline alkaloids (1-11) were isolated from dried leaves of Peumus boldus Mol. by standard chromatographic methods. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analysis, and by comparison with literature data. Compounds isolated in sufficient amount were evaluated for their acetylcholinesterase, and butyrylcholinesterase inhibition activity using Ellman's method. In the prolyl oligopeptidase assay, Z-Gly-Pro-p-nitroanilide was used as substrate. Promising butyrylcholinesterase inhibition activities were demonstrated by two benzylisoquinoline alkaloids, reticuline (8) and N-methylcoclaurine (9), with IC50 values of 33.6 ± 3.0 µM and 15.0 ± 1.4 µM, respectively. Important prolyl oligopeptidase inhibition activities were shown by N-methyllaurotetanine (6) and sinoacutine (4) with IC50 values of 135.4 ± 23.2 µM and 143.1 ± 25.4 µM, respectively. Other tested compounds were considered inactive.

  11. Probing the active sites of butyrylcholinesterase and cholesterol esterase with isomalathion: conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase.

    PubMed

    Doorn, J A; Talley, T T; Thompson, C M; Richardson, R J

    2001-07-01

    Previous work has shown that acetylcholinesterase (AChE), a member of the alpha/beta-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other alpha/beta-hydrolases are stereoselectively inhibited by isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other alpha/beta-hydrolases. Bimolecular rate constants of inhibition (k(i)) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) > or = (1S,3S). Ratios of k(i) values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate constants of reactivation (k(3)) were measured for enzyme inhibited by isomalathion isomers. HBChE, EBChE, and BCholE inactivated by the (1R)-isomers readily reactivated. However, enzymes modified by (1S)-isomalathions were refractory toward reactivation, and k(3) values were not significantly different from zero for HBChE and BCholE treated with the (1S,3S)-isomer. Computer-based docking experiments were performed for BCholE with (1R,3R)- and (1S,3S)-enantiomers. Calculated structures predicted a difference in primary leaving group: diethyl thiosuccinate for (1R,3R)-isomalathion and thiomethyl for the (1S,3S)-isomer. The data demonstrate that the alpha/beta-hydrolases used in this study are stereoselectively inhibited by isomalathion. Furthermore, the results suggest that the mechanistic shift demonstrated to occur for inhibition of AChE by (1R)- versus (1S,3S)-isomers is conserved for butyrylcholinesterase and cholesterol esterase.

  12. Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms

    PubMed Central

    Spieker, Janine; Ackermann, Anica; Salfelder, Anika; Vogel-Höpker, Astrid; Layer, Paul G.

    2016-01-01

    Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein. PMID:27574787

  13. An acetylcholinesterase (AChE) biosensor with enhanced solvent resistance based on chitosan for the detection of pesticides.

    PubMed

    Warner, John; Andreescu, Silvana

    2016-01-01

    Solvent tolerance of immobilized enzymes is important for many biosensing and biotechnological applications. In this paper we report an acetylcholinesterase (AChE) biosensor based on chitosan that exhibits high solvent resistance and enables sensitive detection of pesticides in presence of a high content of organic solvents. The solvent effect was established comparatively for the enzyme immobilized in chitosan and covalently cross-linked with glutaraldehyde. The activity of the immobilized AChE was dependent on the immobilization method and solvent type. The enzyme entrapped in chitosan fully conserved its activity in up to 25% methanol, 15% acetonitrile and 100% cyclohexane while the enzyme cross-linked with glutaraldehyde gradually lost its activity starting at 5% acetonitrile and methanol, and showed variable levels in cyclohexane. The detection limits of the biosensor for paraoxon were: 7.5 nM in 25% methanol, 100 nM in 15% acetonitrile and 2.5 μM in 100% cyclohexane. This study demonstrates that chitosan provides an excellent immobilization environment for AChE biosensors designed to operate in environments containing high amounts of organic solvents. It also highlights the effect of the immobilization material and solvent type on enzyme stability. These findings can enable future selection of the immobilization matrix and solvent type for the development of organic phase enzyme based systems.

  14. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

    PubMed Central

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis. PMID:24675419

  15. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.

    PubMed

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.

  16. Anticancer drugs induce hypomethylation of the acetylcholinesterase promoter via a phosphorylated-p38-DNMT1-AChE pathway in apoptotic hepatocellular carcinoma cells.

    PubMed

    Xi, Qiliang; Gao, Ning; Yang, Yang; Ye, Weiyuan; Zhang, Bo; Wu, Jun; Jiang, Gening; Zhang, Xuejun

    2015-11-01

    Apoptosis, also known as programmed cell death, plays an essential role in eliminating excessive, damaged or harmful cells. Previous work has demonstrated that anticancer drugs induce cell apoptosis by inducing cytotoxicity. In recent years, several reports demonstrated modulated expression of DNA methyltransferases 1 (DNMT1) and acetylcholinesterase (AChE) in a variety of tumors. In this study, we showed that the expression of DNMT1 was decreased and the methylation of CpGs in the promoter of AChE was reduced in anticancer drugs-induced apoptotic hepatocellular carcinoma cells. Silencing of DNMT1 expression by AZA or RNA interference (RNAi) restored AChE production and inhibition of AChE expression by RNAi protected HCC cells from anticancer drugs-induced apoptosis. Furthermore, we demonstrated that the regulation of AChE by DNMT1 was involved in the phosphorylated p38 pathway in anticancer drugs-induced apoptosis. In addition, immunohistochemical staining showed that P-p38, DNMT1 and AChE were aberrantly expressed in a subset of HCC tumors. Taken together, we demonstrated the regulation of AChE by DNMT1 and further, we found that this regulation was involved in the phosphorylated p38 pathway in anticancer drugs-induced apoptosis.

  17. Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of 4-amino-2, 3-diaryl-5, 6, 7, 8-tetrahydrofuro(and thieno)[2, 3-b]-quinolines, and 4-amino-5, 6, 7, 8, 9-pentahydro-2, 3-diphenylcyclohepta[e]furo(and thieno)-[2, 3-b]pyridines.

    PubMed

    Marco, José L; De Los Ríos, Cristóbal; Carreiras, María C; Baños, Josep E; Badia, Albert; Vivas, Nuria M

    2002-07-01

    The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition activities of a series of 4-amino-2, 3-diaryl-5, 6, 7, 8-tetrahydrofuro[2, 3-b]quinolines (10-12)/4-amino-5, 6, 7, 8-tetrahydro-2, 3-diphenylthieno[2, 3-b]quinoline (14) and 4-amino-5, 6, 7, 8, 9-pentahydro-2, 3-diphenylcyclohepta[e]furo[2, 3-b]pyridine (13)/4-amino-5, 6, 7, 8, 9-pentahydro-2, 3-phenylcyclohepta[e]thieno[2, 3-b]pyridine (15) are described. These compounds are tacrine (THA) analogues which have been prepared either from readily available 2-amino-3-cyano-4, 5-diarylfurans (16-18) or from 2-amino-3-cyano-4, 5-diphenylthiophene (19), via Friedländer condensation with cyclohexanone or cycloheptanone. These compounds are competitive inhibitors for acetylcholinesterase, the more potent being compound (13) which is three-fold less active than tacrine. The butyrylcholinesterase inhibition activity is significant only in compounds 10 and133, which are ten-fold less active than tacrine. It is found that the products 11 and 12 strongly inhibit acetylcholinesterase, and show excellent selectivity regarding butyrylcholinesterase.

  18. Effect of acetylcholinesterase (AChE) point-of-care testing in OP poisoning on knowledge, attitudes and practices of treating physicians in Sri Lanka

    PubMed Central

    2014-01-01

    Background Toxicology and Emergency medicine textbooks recommend measurement of acetylcholinesterase (AChE) in all symptomatic cases of organophosphorus (OP) poisoning but laboratory facilities are limited in rural Asia. The accuracy of point-of-care (POC) acetylcholinesterase testing has been demonstrated but it remains to be shown whether results would be valued by clinicians. This study aims to assess the effect of seeing AChE POC test results on the knowledge, attitudes and practices of doctors who frequently manage OP poisoning. Methods We surveyed 23 clinicians, who had different levels of exposure to seeing AChE levels in OP poisoned patients, on a) knowledge of OP poisoning and biomarker interpretation, b) attitudes towards AChE in guiding poison management, oxime therapy and discharge decisions, and c) practices of ordering AChE in poisoning scenarios. Results An overall high proportion of doctors valued the test (68-89%). However, we paradoxically found that doctors who were more experienced in seeing AChE results valued the test less. Lower proportions valued the test in guidance of acute poisoning management (50%, p = 0.015) and guidance of oxime therapy (25%, p = 0.008), and it was apparent it would not generally be used to facilitate early discharge. The highest proportion of respondents valued it on admission (p < 0.001). A lack of correlation of test results with the clinical picture, and a perception that the test was a waste of money when compared to clinical observation alone were also comments raised by some of the respondents. Greater experience with seeing AChE test results was associated with increased knowledge (p = 0.034). However, a disproportionate lack of knowledge on interpretation of biomarkers and the pharmacology of oxime therapy (12-50%) was noted, when compared with knowledge on the mechanism of OP poisoning and management (78-90%). Conclusions Our findings suggest an AChE POC test may not be valued by rural doctors. The practical

  19. Acetylcholinesterase (AChE)--amyloid-beta-peptide complexes in Alzheimer's disease. the Wnt signaling pathway.

    PubMed

    Inestrosa, Nibaldo C; Urra, Soledad; Colombres, Marcela

    2004-11-01

    Alzheimer's disease (AD) is characterized by selective neuronal cell death, which is probably caused by amyloid beta-peptide (Abeta) oligomers and fibrils. We have found that acetylcholinesterase (AChE), a senile plaque component, increases amyloid fibril assembly with the formation of highly toxic complexes (Abeta-AChE). The neurotoxic effect induced by Abeta-AChE complexes was higher than that induced by the Abeta peptide alone as shown both in vitro (hippocampal neurons) and in vivo (rats injected with Abeta peptide in the dorsal hippocampus). Interestingly, treatment with Abeta-AChE complexes decreases the cytoplasmic beta-catenin level, a key component of Wnt signaling. Conversely, the activation of this signaling pathway by Wnt-3a promotes neuronal survival and rescues changes in Wnt components (activation or subcellular localization). Moreover Frzb-1, a Wnt antagonist reverses the Wnt-3a neuroprotection effect against Abeta neurotoxicity. Compounds that mimic the Wnt signaling or modulate the cross-talking with this pathway could be used as neuroprotective agents for therapeutic strategies in AD patients.

  20. Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

    PubMed Central

    Blohberger, J; Kunz, L; Einwang, D; Berg, U; Berg, D; Ojeda, S R; Dissen, G A; Fröhlich, T; Arnold, G J; Soreq, H; Lara, H; Mayerhofer, A

    2015-01-01

    Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel

  1. Acetylcholinesterase and butyrylcholinesterase inhibition of ethanolic extract and monoterpenes from Pimpinella anisoides V Brig. (Apiaceae).

    PubMed

    Menichini, Federica; Tundis, Rosa; Loizzo, Monica R; Bonesi, Marco; Marrelli, Mariangela; Statti, Giancarlo A; Menichini, Francesco; Conforti, Filomena

    2009-07-01

    Ethanolic extract from the fruits of Pimpinella anisoides, an aromatic plant and a spice, exhibited activity against AChE and BChE, with IC(50) values of 227.5 and 362.1 microg/ml, respectively. The most abundant constituents of the extract were trans-anethole, (+)-limonene and (+)-sabinene. trans-Anethole exhibited the highest activity against AChE and BChE with IC(50) values of 134.7 and 209.6 microg/ml, respectively. The bicyclic monoterpene (+)-sabinene exhibited a promising activity against AChE (IC(50) of 176.5 microg/ml) and BChE (IC(50) of 218.6 microg/ml).

  2. Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?

    PubMed

    Fossati, Sara Maria; Candiani, Simona; Nödl, Marie-Therese; Maragliano, Luca; Pennuto, Maria; Domingues, Pedro; Benfenati, Fabio; Pestarino, Mario; Zullo, Letizia

    2015-08-01

    Acetylcholinesterase (ACHE) is a glycoprotein with a key role in terminating synaptic transmission in cholinergic neurons of both vertebrates and invertebrates. ACHE is also involved in the regulation of cell growth and morphogenesis during embryogenesis and regeneration acting through its non-cholinergic sites. The mollusk Octopus vulgaris provides a powerful model for investigating the mechanisms underlying tissue morphogenesis due to its high regenerative power. Here, we performed a comparative investigation of arm morphogenesis during adult arm regeneration and embryonic arm development which may provide insights on the conserved ACHE pathways. In this study, we cloned and characterized O. vulgaris ACHE, finding a single highly conserved ACHE hydrophobic variant, characterized by prototypical catalytic sites and a putative consensus region for a glycosylphosphatidylinositol (GPI)-anchor attachment at the COOH-terminus. We then show that its expression level is correlated to the stage of morphogenesis in both adult and embryonic arm. In particular, ACHE is localized in typical neuronal sites when adult-like arm morphology is established and in differentiating cell locations during the early stages of arm morphogenesis. This possibility is also supported by the presence in the ACHE sequence and model structure of both cholinergic and non-cholinergic sites. This study provides insights into ACHE conserved roles during processes of arm morphogenesis. In addition, our modeling study offers a solid basis for predicting the interaction of the ACHE domains with pharmacological blockers for in vivo investigations. We therefore suggest ACHE as a target for the regulation of tissue morphogenesis.

  3. Dual Binding Site and Selective Acetylcholinesterase Inhibitors Derived from Integrated Pharmacophore Models and Sequential Virtual Screening

    PubMed Central

    Gupta, Shikhar; Mohan, C. Gopi

    2014-01-01

    In this study, we have employed in silico methodology combining double pharmacophore based screening, molecular docking, and ADME/T filtering to identify dual binding site acetylcholinesterase inhibitors that can preferentially inhibit acetylcholinesterase and simultaneously inhibit the butyrylcholinesterase also but in the lesser extent than acetylcholinesterase. 3D-pharmacophore models of AChE and BuChE enzyme inhibitors have been developed from xanthostigmine derivatives through HypoGen and validated using test set, Fischer's randomization technique. The best acetylcholinesterase and butyrylcholinesterase inhibitors pharmacophore hypotheses Hypo1_A and Hypo1_B, with high correlation coefficient of 0.96 and 0.94, respectively, were used as 3D query for screening the Zinc database. The screened hits were then subjected to the ADME/T and molecular docking study to prioritise the compounds. Finally, 18 compounds were identified as potential leads against AChE enzyme, showing good predicted activities and promising ADME/T properties. PMID:25050335

  4. Assessment of acetylcholinesterase and butyrylcholinesterase activities in blood plasma of agriculture workers

    PubMed Central

    Dhananjayan, V.; Ravichandran, B.; Anitha, N.; Rajmohan, H. R.

    2012-01-01

    Background: Cholinesterase determination indicates whether the person has been under pesticide exposure is not. It is recommended that the worker′s cholinesterase level should be assessed for workers at a pesticide applied region. Hence, cholinesterase activities in blood samples of agricultural workers exposed to vegetables and grape cultivation with age matched, unexposed workers, who never had any exposure to pesticides, were estimated. Methods: The detailed occupational history and lifestyle characters were obtained by questionnaire. Cholinesterase activity was determined by the method of Ellman as modified by Chambers and Chambers. Results: AChE was ranging from 1.65 to 3.54μmoles/min/ml in exposed subjects where as it was ranged from 2.22 to 3.51μmoles/min/ml in control subjects. BChE activity was ranging from 0.16 to 5.2μmoles/min/ml among exposed subjects, where as it was ranged from 2.19 to 5.06μmoles/min/ml in control subjects. The results showed statistically significant reduction in enzyme activities (AChE 14%; BChE 56%) among exposed subjects. Conclusion: It was concluded that the reduction in cholinesterase activity may lead to varieties of effects. Hence it is compulsory to use protective gadgets during pesticide spray. Further a continuous biomonitoring study is recommended to assess pesticide exposure. PMID:23776322

  5. 2-Benzoyl-6-benzylidenecyclohexanone analogs as potent dual inhibitors of acetylcholinesterase and butyrylcholinesterase.

    PubMed

    Leong, Sze Wei; Abas, Faridah; Lam, Kok Wai; Shaari, Khozirah; Lajis, Nordin H

    2016-08-15

    In the present study, a series of 2-benzoyl-6-benzylidenecyclohexanone analogs have been synthesized and evaluated for their anti-cholinesterase activity. Among the forty-one analogs, four compounds (38, 39, 40 and 41) have been identified as lead compounds due to their highest inhibition on both AChE and BChE activities. Compounds 39 and 40 in particular exhibited highest inhibition on both AChE and BChE with IC50 values of 1.6μM and 0.6μM, respectively. Further structure-activity relationship study suggested that presence of a long-chain heterocyclic in one of the rings played a critical role in the dual enzymes' inhibition. The Lineweaver-Burk plots and docking results suggest that both compounds could simultaneously bind to the PAS and CAS regions of the enzyme. ADMET analysis further confirmed the therapeutic potential of both compounds based upon their high BBB-penetrating. Thus, 2-benzoyl-6-benzylidenecyclohexanone containing long-chain heterocyclic amine analogs represent a new class of cholinesterase inhibitor, which deserve further investigation for their development into therapeutic agents for cognitive diseases such as Alzheimer.

  6. Acetylcholinesterase Inhibitors (AChEI's) for the treatment of visual hallucinations in schizophrenia: A review of the literature

    PubMed Central

    2010-01-01

    Background Visual hallucinations occur in various neurological diseases, but are most prominent in Lewy body dementia, Parkinson's disease and schizophrenia. The lifetime prevalence of visual hallucinations in patients with schizophrenia is much more common than conventionally thought and ranges from 24% to 72%. Cortical acetylcholine (ACh) depletion has been associated with visual hallucinations; the level of depletion being related directly to the severity of the symptoms. Current understanding of neurobiological visual processing and research in diseases with reduced cholinergic function, suggests that AChEI's may prove beneficial in treating visual hallucinations. This offers the potential for targeted drug therapy of clinically symptomatic visual hallucinations in patients with schizophrenia using acetylcholinesterase inhibition. Methods A systematic review was carried out investigating the evidence for the effects of AChEI's in treating visual hallucinations in Schizophrenia. Results No evidence was found relating to the specific role of AChEI's in treating visual hallucinations in this patient group. Discussion Given the use of AChEI's in targeted, symptom specific treatment in other neuropsychiatric disorders, it is surprising to find no related literature in schizophrenia patients. The use of AChEI's in schizophrenia has investigated effects on cognition primarily with non cognitive effects measured more broadly. Conclusions We would suggest that more focused research into the effects of AChEI's on positive symptoms of schizophrenia, specifically visual hallucinations, is needed. PMID:20822517

  7. In vitro reactivation of sarin-inhibited human acetylcholinesterase (AChE) by bis-pyridinium oximes connected by xylene linkers.

    PubMed

    Acharya, Jyotiranjan; Dubey, Devendra Kumar; Srivastava, Ashish Kumar; Raza, Syed Kalbey

    2011-02-01

    A series of bis-pyridinium oximes connected by xylene linkers were synthesized and their in vitro reactivation potential was evaluated against human acetylcholinesterase (hAChE) inhibited by nerve agent sarin and the data were compared with 2-PAM and obidoxime. Among the synthesized compounds, N,N'-p-xylene-bis-[(2,2'-hydroxyiminomethyl)pyridinium] dibromide (3c) was found to be the most potent reactivator for hAChE inhibited by sarin. The oxime 3c exhibited 45% regeneration of inhibited hAChE, in comparison to 34% and 24% regeneration by 2-PAM and obidoxime, respectively, at a concentration of 10(-3) M within 10 min. The higher reactivation efficacies of these oximes were attributed to their acid dissociation constants (pKa). The pKa values of all the oximes were determined spectrophotometrically and correlated with their observed reactivation potential. This method involving the in vitro reactivation of inhibited hAChE may be useful for the screening of new oximes as reactivators.

  8. Fluorescence Quenching Determination of Uranium (VI) Binding Properties by Two Functional Proteins: Acetylcholinesterase (AChE) and Vitellogenin (Vtg).

    PubMed

    Coppin, Frédéric; Michon, Jérôme; Garnier, Cédric; Frelon, Sandrine

    2015-05-01

    The interactions between uranium and two functional proteins (AChE and Vtg) were investigated using fluorescence quenching measurements. The combined use of a microplate spectrofluorometer and logarithmic additions of uranium into protein solutions allowed us to define the fluorescence quenching over a wide range of [U]/[Pi] ratios (from 1 to 3235) at physiologically relevant conditions of pH. Results showed that fluorescence from the two functional proteins was quenched by UO2 (2+). Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of proteins, i.e. binding constants and binding sites densities, were determined using classic fluorescence quenching methods and curve-fitting software (PROSECE). It was demonstrated that in our test conditions, the protein complexation by uranium could be simulated by two specific sites (L1 and L2). The obtained complexation constant values are log K1 = 5.7 (±1.0), log K2 = 4.9 (±1.1); L1 = 83 (±2), L2 = 2220 (±150) for U(VI) - Vtg and log K1 = 8.1 (±0.9), log K2 = 6.6 (±0.5), L1 = 115 (±16), L2 = 530 (±23) for U(VI)-AChE (Li is expressed in mol/mol of protein).

  9. Structural modifications of 4-aryl-4-oxo-2-aminylbutanamides and their acetyl- and butyrylcholinesterase inhibitory activity. Investigation of AChE-ligand interactions by docking calculations and molecular dynamics simulations.

    PubMed

    Vitorović-Todorović, Maja D; Koukoulitsa, Catherine; Juranić, Ivan O; Mandić, Ljuba M; Drakulić, Branko J

    2014-06-23

    Congeneric set of thirty-eight 4-aryl-4-oxo-2-(N-aryl/cycloalkyl)butanamides has been designed, synthesized and evaluated for acetyl- and butyrylcholinesterase inhibitory activity. Structural variations included cycloalkylamino group attached to C2 position of butanoyl moiety, and variation of amido moiety of molecules. Twelve compounds, mostly piperidino and imidazolo derivatives, inhibited AChE in low micromolar range, and were inactive toward BChE. Several N-methylpiperazino derivatives showed inhibition of BChE in low micromolar or submicromolar concentrations, and were inactive toward AChE. Therefore, the nature of the cycloalkylamino moiety governs the AChE/BChE selectivity profile of compounds. The most active AChE inhibitor showed mixed-type inhibition modality, indicating its binding to free enzyme and to enzyme-substrate complex. Thorough docking calculations of the seven most potent AChE inhibitors from the set, showed that the hydrogen bond can be formed between amide -NH- moiety of compounds and -OH group of Tyr 124. The 10 ns unconstrained molecular dynamic simulation of the AChE-compound 18 complex shows that this interaction is the most persistent. This is, probably, the major anchoring point for the binding.

  10. Synthesis, pharmacological assessment, and molecular modeling of acetylcholinesterase/butyrylcholinesterase inhibitors: effect against amyloid-β-induced neurotoxicity.

    PubMed

    Silva, Daniel; Chioua, Mourad; Samadi, Abdelouahid; Agostinho, Paula; Garção, Pedro; Lajarín-Cuesta, Rocío; de Los Ríos, Cristobal; Iriepa, Isabel; Moraleda, Ignacio; Gonzalez-Lafuente, Laura; Mendes, Eduarda; Pérez, Concepción; Rodríguez-Franco, María Isabel; Marco-Contelles, José; Carmo Carreiras, M

    2013-04-17

    The synthesis, molecular modeling, and pharmacological analysis of phenoxyalkylamino-4-phenylnicotinates (2-7), phenoxyalkoxybenzylidenemalononitriles (12, 13), pyridonepezils (14-18), and quinolinodonepezils (19-21) are described. Pyridonepezils 15-18 were found to be selective and moderately potent regarding the inhibition of hAChE, whereas quinolinodonepezils 19-21 were found to be poor inhibitors of hAChE. The most potent and selective hAChE inhibitor was ethyl 6-(4-(1-benzylpiperidin-4-yl)butylamino)-5-cyano-2-methyl-4-phenylnicotinate (18) [IC(50) (hAChE) = 0.25 ± 0.02 μM]. Pyridonepezils 15-18 and quinolinodonepezils 20-21 are more potent selective inhibitors of EeAChE than hAChE. The most potent and selective EeAChE inhibitor was ethyl 6-(2-(1-benzylpiperidin-4-yl)ethylamino)-5-cyano-2-methyl-4-phenylnicotinate (16) [IC(50) (EeAChE) = 0.0167 ± 0.0002 μM], which exhibits the same inhibitory potency as donepezil against hAChE. Compounds 2, 7, 13, 17, 18, 35, and 36 significantly prevented the decrease in cell viability caused by Aβ(1-42). All compounds were effective in preventing the enhancement of AChE activity induced by Aβ(1-42). Compounds 2-7 caused a significant reduction whereas pyridonepezils 17 and 18, and compound 16 also showed some activity. The pyrazolo[3,4-b]quinolines 36 and 38 also prevented the upregulation of AChE induced by Aβ(1-42). Compounds 2, 7, 12, 13, 17, 18, and 36 may act as antagonists of voltage sensitive calcium channels, since they significantly prevented the Ca(2+) influx evoked by KCl depolarization. Docking studies show that compounds 16 and 18 adopted different orientations and conformations inside the active-site gorges of hAChE and hBuChE. The structural and energetic features of the 16-AChE and 18-AChE complexes compared to the 16-BuChE and 18-BuChE complexes account for a higher affinity of the ligand toward AChE. The present data indicate that compounds 2, 7, 17, 18, and 36 may represent attractive multipotent

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

    PubMed

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

    2001-11-01

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

  12. Acetylcholinesterase Inhibitors (AChEI's) for the treatment of visual hallucinations in schizophrenia: a case report

    PubMed Central

    2010-01-01

    Background Visual hallucinations are commonly seen in various neurological and psychiatric disorders including schizophrenia. Current models of visual processing and studies in diseases including Parkinsons Disease and Lewy Body Dementia propose that Acetylcholine (Ach) plays a pivotal role in our ability to accurately interpret visual stimuli. Depletion of Ach is thought to be associated with visual hallucination generation. AchEI's have been used in the targeted treatment of visual hallucinations in dementia and Parkinson's Disease patients. In Schizophrenia, it is thought that a similar Ach depletion leads to visual hallucinations and may provide a target for drug treatment Case Presentation We present a case of a patient with Schizophrenia presenting with treatment resistant and significantly distressing visual hallucinations. After optimising treatment for schizophrenia we used Rivastigmine, an AchEI, as an adjunct to treat her symptoms successfully. Conclusions This case is the first to illustrate this novel use of an AchEI in the targeted treatment of visual hallucinations in a patient with Schizophrenia. Targeted therapy of this kind can be considered in challenging cases although more evidence is required in this field. PMID:20822516

  13. Reduced Acetylcholine Receptor Density, Morphological Remodeling, and Butyrylcholinesterase Activity Can Sustain Muscle Function in Acetylcholinesterase Knockout Mice

    DTIC Science & Technology

    2004-09-01

    superior catalytic activity for mouse chow and water ad libitum and were 53 + 4 ACh hydrolysis, AChE is the dominant enzyme, days old at the time of...were analyzed using a two-tailed gain, 1593; scan time , 16 s). Endplates in the same Student’s t- test , whereas comparisons between three focal plane...amplitudes and prolonged knockout mice , this enzyme appears to exert a prom- rise and relaxation times similar to those observed in inent role in

  14. Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase

    SciTech Connect

    Li Bin; Duysen, Ellen G.; Poluektova, Larisa Y.; Murrin, L. Charles . E-mail: cmurrin@unmc.edu; Lockridge, Oksana . E-mail: olockrid@unmc.edu

    2006-07-15

    Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months in plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC 3.1.1.8) in motor endplates.

  15. Alkaloids from Habranthus tubispathus and H. jamesonii, two amaryllidaceae with acetyl- and butyrylcholinesterase inhibition activity.

    PubMed

    Cavallaro, Valeria; Alza, Natalia P; Murray, María G; Murray, Ana P

    2014-02-01

    Alzheimer's disease (AD) is a neurodegenerative disorder associated with memory impairment and cognitive deficit. Most of the drugs currently available for the treatment of AD are acetylcholinesterase (AChE) inhibitors. Plants of the Amaryllidaceae family are known to synthesize alkaloids, which have shown AChE inhibitory activity. Habranthus tubispathus and H. jamesonii are two Amaryllidaceae that can be found growing wild to the southwest of Buenos Aires in Argentina. Acetyl- and butyrylcholinesterase inhibition was observed for the extracts obtained from bulbs of H. tubispathus and bulbs and aerial parts of H. jamesonii. The strongest cholinesterase inhibition was observed for the alkaloid extract obtained from the aerial parts for H. jamesonii (AChE IC50 = 0.7 microg/mL; BChE IC50 = 6.7 microg/mL). The AChE inhibition observed for H. jamesonii could be explained by the presence of galanthamine and sanguinine, two potent AChE inhibitors. The levels of lycorine and hippeastidine, moderate AChE inhibitors, observed in the bulbs of H. tubispathus could be responsible for the significant AChE inhibition observed. The alkaloids present in these Amaryllidaceae were identified by means of GC-MS analysis. In the case of H. tubispathus, hippeastidine and 3-O-demethylhippeastidine, were isolated and completely characterized by 1H and 13C NMR spectroscopy.

  16. Does time difference of the acetylcholinesterase (AChE) inhibition in different tissues exist? A case study of zebra fish (Danio rerio) exposed to cadmium chloride and deltamethrin.

    PubMed

    Zhang, Tingting; Yang, Meiyi; Pan, Hongwei; Li, Shangge; Ren, Baigang; Ren, Zongming; Xing, Na; Qi, Luhuizi; Ren, Qing; Xu, Shiguo; Song, Jie; Ma, Jingchun

    2017-02-01

    In order to illustrate time difference in toxic effects of cadmium chloride (CdCl2) and deltamethrin (DM), AChE activities were measured in different tissues, liver, muscle, brain, and gill, of Zebra fish (Danio rerio) across different concentrations in this research. The average AChE activity decreased comparing to 0.0 TU with DM (82.81% in 0.1 TU, 56.14% in 1.0 TU and 44.68% in 2.0 TU) and with CdCl2 (74.68% in 0.1 TU, 52.05% in 1.0 TU and 50.14% in 2.0 TU) showed an overall decrease with the increase of exposure concentrations. According to Self-Organizing Map (SOM), the AChE activities were characterized in relation with experimental conditions, showing an inverse relationship with exposure time. As the exposure time was longer, the AChE activities were correspondingly lower. The AChE inhibition showed time delay in sublethal treatments (0.1 TU) in different tissues: the AChE was first inhibited in brain by chemicals followed by gill, muscle and liver (brain > gill > muscle > liver). The AChE activity was almost inhibited synchronously in higher environmental stress (1.0 TU and 2.0 TU). As the AChE inhibition can induce abnormal of behavior movement, these results will be helpful to the mechanism of stepwise behavior responses according to the time difference in different tissues rather than the whole body AChE activity.

  17. Schwann cells sense and control acetylcholine spillover at the neuromuscular junction by α7 nicotinic receptors and butyrylcholinesterase.

    PubMed

    Petrov, Konstantin A; Girard, Emmanuelle; Nikitashina, Alexandra D; Colasante, Cesare; Bernard, Véronique; Nurullin, Leniz; Leroy, Jacqueline; Samigullin, Dmitry; Colak, Omer; Nikolsky, Evgenii; Plaud, Benoit; Krejci, Eric

    2014-09-03

    Terminal Schwann cells (TSCs) are key components of the mammalian neuromuscular junction (NMJ). How the TSCs sense the synaptic activity in physiological conditions remains unclear. We have taken advantage of the distinct localization of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at the NMJ to bring out the function of different ACh receptors (AChRs). AChE is clustered by the collagen Q in the synaptic cleft and prevents the repetitive activation of muscle nicotinic AChRs. We found that BChE is anchored at the TSC by a proline-rich membrane anchor, the small transmembrane protein anchor of brain AChE. When BChE was specifically inhibited, ACh release was significant depressed through the activation of α7 nAChRs localized on the TSC and activated by the spillover of ACh. When both AChE and BChE were inhibited, the spillover increased and induced a dramatic reduction of ACh release that compromised the muscle twitch triggered by the nerve stimulation. α7 nAChRs at the TSC may act as a sensor for spillover of ACh adjusted by BChE and may represent an extrasynaptic sensor for homeostasis at the NMJ. In myasthenic rats, selective inhibition of AChE is more effective in rescuing muscle function than the simultaneous inhibition of AChE and BChE because the concomitant inhibition of BChE counteracts the positive action of AChE inhibition. These results show that inhibition of BChE should be avoided during the treatment of myasthenia and the pharmacological reversal of residual curarization after anesthesia.

  18. Biochemical effects of glyphosate based herbicide, Excel Mera 71 on enzyme activities of acetylcholinesterase (AChE), lipid peroxidation (LPO), catalase (CAT), glutathione-S-transferase (GST) and protein content on teleostean fishes.

    PubMed

    Samanta, Palas; Pal, Sandipan; Mukherjee, Aloke Kumar; Ghosh, Apurba Ratan

    2014-09-01

    Effects of glyphosate based herbicide, Excel Mera 71 at a dose of 17.20mg/l on enzyme activities of acetylcholinesterase (AChE), lipid peroxidation (LPO), catalase (CAT), glutathione-S-transferase (GST) and protein content were measured in different tissues of two Indian air-breathing teleosts, Anabas testudineus (Bloch) and Heteropneustes fossilis (Bloch) during an exposure period of 30 days under laboratory condition. AChE activity was significantly increased in all the investigated tissues of both fish species and maximum elevation was observed in brain of H. fossilis, while spinal cord of A. testudineus showed minimum increment. Fishes showed significant increase LPO levels in all the tissues; highest was observed in gill of A. testudineus but lowest LPO level was observed in muscle of H. fossilis. CAT was also enhanced in both the fishes, while GST activity in liver diminished substantially and minimum was observed in liver of A. testudineus. Total protein content showed decreased value in all the tissues, maximum reduction was observed in liver and minimum in brain of A. testudineus and H. fossilis respectively. The results indicated that Excel Mera 71 caused serious alterations in the enzyme activities resulting into severe deterioration of fish health; so, AChE, LPO, CAT and GST can be used as suitable indicators of herbicidal toxicity.

  19. Synthesis and in vitro kinetic study of novel mono-pyridinium oximes as reactivators of organophosphorus (OP) inhibited human acetylcholinesterase (hAChE).

    PubMed

    Valiveti, Aditya Kapil; Bhalerao, Uma M; Acharya, Jyotiranjan; Karade, Hitendra N; Gundapu, Raviraju; Halve, Anand K; Kaushik, Mahabir Parshad

    2015-07-25

    A series of mono pyridinium oximes linked with arenylacetamides as side chains were synthesized and their in vitro reactivation potential was evaluated against human acetylcholinesterase (hAChE) inhibited by organophosphorus inhibitors (OP) such as sarin, VX and tabun. The reactivation data of the synthesized compounds were compared with those obtained with standard reactivators such as 2-PAM and obidoxime. The dissociation constant (KD) and specific reactivity (kr) of the oximes were also determined by performing reactivation kinetics against OP inhibited hAChE. Among the synthesized compounds, oximes 1-(2-(4-cyanophenylamino)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium chloride (12a) and 4-((hydroxyimino)methyl)-1-(2-(4-methoxyphenylamino)-2-oxoethyl)pyridinium chloride (2a) were found most potent reactivators for hAChE inhibited by sarin. In case of VX inhibited hAChE majority of the oximes have shown good reactivation efficacies. Among these oximes 1-(2-(benzylamino)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium chloride (18a), 4-((hydroxyimino)methyl)-1-(2-(4-(methoxycarbonyl)phenylamino)-2-oxoethyl)pyridinium-chloride (14a) and 12a were found to surpass the reactivation potential of 2-PAM and obidoxime. However, the synthesized oximes showed marginal reactivation efficacies in case of tabun inhibited hAChE. The pKa value of the oximes were determined and correlated with their observed reactivation potential.

  20. Functional Analysis and Molecular Docking studies of Medicinal Compounds for AChE and BChE in Alzheimer’s Disease and Type 2 Diabetes Mellitus

    PubMed Central

    Kaladhar, Dowluru SVGK; Yarla, Nagendra Sastry; Anusha, N.

    2013-01-01

    Acetylcholinesterase and Butyrylcholinesterase share unravelling link with components of metabolic syndromes that’s characterised by low levels of HDL cholesterol, obesity, high fast aldohexose levels, hyper-trigliceridaemia and high blood pressure, by regulation of cholinergic transmission and therefore the enzyme activity within a living system. The phosphomotifs associated with amino acid and tyrosine binding motifs in AChE and BChE were known to be common. Phylogenetic tree was constructed to these proteins usinf UPGMA and Maximum Likelihood methods in MEGA software has shown interaction of AChE and BChE with ageing diseases like Alzheimer’s disease and Diabetes. AChE has shown closely related to BChE, retinol dehydrogenase and β-polypeptide. The present studies is also accomplished that AChE, BChE, COLQ, HAND1, APP, NLGN2 and NGF proteins has interactions with diseases such as Alzheimer’s and D2M using Pathwaylinker and STRING. Medicinal compounds like Ortho-7, Dibucaine and HI-6 are predicted as good targets for modeled AChE and BChE proteins based on docking studies. Hence perceptive studies of cholinesterase structure and the biological mechanisms of inhibition are necessary for effective drug development. PMID:23936743

  1. Acute toxicity of a commercial glyphosate formulation on European sea bass juveniles (Dicentrarchus labrax L.): gene expressions of heme oxygenase-1 (ho-1), acetylcholinesterase (AChE) and aromatases (cyp19a and cyp19b).

    PubMed

    Prevot-D'Alvise, N; Richard, S; Coupé, S; Bunet, R; Grillasca, J P

    2013-12-31

    Acute toxicity of Roundup, a commercial glyphosate--based herbicide, was evaluated in a teleost marine fish, the European sea bass, after 96 h of exposure. The LC50 96-h value of Roundup was 529 mg/L. Juveniles (Dicentrarchus labrax L.) were exposed to a sublethal concentration (35% of the LC50, i.e. 193 mg/L) of Roundup for 96-h. The study of heme oxygenase-1 (ho-1) gene expression was performed in four tissues (liver, gills, brain and gonads) and highlighted the disruption of antioxidant defence system. Results showed that ho-1 mRNA levels in liver and gills significantly decreased (p<0.001 and p<0.01 respectively) in fish exposed to 193 mg/L of Roundup, whereas in brain and gonads, ho-1 mRNA level was not altered. The analysis of acetylcholinesterase expression was used to evaluate the overall neurotoxicity of the herbicide and aromatase genes to assess the alteration of the endocrine system. Results showed that AChE and cyp19b gene transcriptions significantly increased (p<0.01) in brain of sea bass, whereas aromatase gene expression (cyp19a) in gonads was not significantly altered. Our results showed complex tissue-specific transcriptional responses after 96 h of exposure to a sublethal concentration. All these disruptions confirmed the deleterious effects of this glyphosate-based herbicide in a marine species.

  2. Synthesis and in-vitro reactivation screening of imidazolium aldoximes as reactivators of sarin and VX-inhibited human acetylcholinesterase (hAChE).

    PubMed

    Sharma, Rahul; Gupta, Bhanushree; Sahu, Arvind Kumar; Acharya, Jyotiranjan; Satnami, Manmohan L; Ghosh, Kallol K

    2016-11-25

    Post-treatment of organophosphate (OP) poisoning involves the application of oxime reactivator as an antidote. Structurally different oximes are widely studied to examine their kinetic and mechanistic behavior against OP-inhibited cholinesterase enzyme. A series of structurally related 1,3-disubstituted-2-[(hydroxyiminomethyl)alkyl]imidazolium halides (5a-5e, 9a-9c) were synthesized and further evaluated for their in-vitro reactivation ability to reactivate sarin- and VX-inhibited human acetylcholinesterase (hAChE). The observed results were compared with the reactivation efficacy of standard reactivators; 2-PAM, obidoxime and HI-6. Amongst the synthesized oximes, 5a, 9a and 9b were found to be most potent reactivators against sarin-inhibited hAChE while in case of VX only 9a exhibited comparable reactivity with 2-PAM. Incorporation of pyridinium ring to the imidazole ring resulted in substantial increase in the reactivation strength of prepared reactivator. Physicochemical properties of synthesized reactivators have also been evaluated.

  3. Virtual Screening of Acetylcholinesterase Inhibitors Using the Lipinski's Rule of Five and ZINC Databank

    PubMed Central

    Nogara, Pablo Andrei; Saraiva, Rogério de Aquino; Caeran Bueno, Diones; Lissner, Lílian Juliana; Lenz Dalla Corte, Cristiane; Braga, Marcos M.; Rosemberg, Denis Broock; Rocha, João Batista Teixeira

    2015-01-01

    Alzheimer's disease (AD) is a progressive and neurodegenerative pathology that can affect people over 65 years of age. It causes several complications, such as behavioral changes, language deficits, depression, and memory impairments. One of the methods used to treat AD is the increase of acetylcholine (ACh) in the brain by using acetylcholinesterase inhibitors (AChEIs). In this study, we used the ZINC databank and the Lipinski's rule of five to perform a virtual screening and a molecular docking (using Auto Dock Vina 1.1.1) aiming to select possible compounds that have quaternary ammonium atom able to inhibit acetylcholinesterase (AChE) activity. The molecules were obtained by screening and further in vitro assays were performed to analyze the most potent inhibitors through the IC50 value and also to describe the interaction models between inhibitors and enzyme by molecular docking. The results showed that compound D inhibited AChE activity from different vertebrate sources and butyrylcholinesterase (BChE) from Equus ferus (EfBChE), with IC50 ranging from 1.69 ± 0.46 to 5.64 ± 2.47 µM. Compound D interacted with the peripheral anionic subsite in both enzymes, blocking substrate entrance to the active site. In contrast, compound C had higher specificity as inhibitor of EfBChE. In conclusion, the screening was effective in finding inhibitors of AChE and BuChE from different organisms. PMID:25685814

  4. Bone status of adult female butyrylcholinesterase gene-deficient mice.

    PubMed

    Haupt, Malte; Kauschke, Vivien; Sender, Jonas; Kampschulte, Marian; Kovtun, Anna; Dürselen, Lutz; Heiss, Christian; Lips, Katrin Susanne

    2015-11-01

    Butyrylcholinesterase (BChE) degrades acetylcholine in addition to acetylcholinesterase (AChE) which is involved in embryonic development of limbs. Since BChE is expressed by osteoblast-like cells we asked whether it is functional in adult bone remodeling. We addressed this issue by analyzing BChE gene-deficient mice (BChE-KO). Bones were extracted from 16-week old female BChE-KO and corresponding wild type mice (WT). Femoral bones were used for biomechanical testing and μCT evaluation of cancellous and cortical bone. Also vertebrae Th12 and L1 were investigated with μCT while L3 was used for tartrate-resistant acidic phosphatase (TRAP) histomorphometry and Th10 for gene expression analysis by means of real-time RT-PCR. BChE-KO did not reveal significant differences in biomechanical bone strength and bone mineral density determined by μCT. Microarchitecture of cancellous and cortical bone showed an increase in μCT parameters like trabecular thickness, trabecular separation, and relative cortical bone area of femoral BChE-KO bone compared to WT. In vertebrae no changes of microstructure and mRNA expression were detected. However, osteoclast histomorphometry with TRAP stained sections demonstrated a significant increase in relative osteoclast number. In conclusion, in adult murine bone the role of BChE is limited to bone specific changes in microarchitecture and to an increase in relative number of bone resorbing osteoclasts whereas the main collagen resorbing enzyme Cathepsin-K (CtsK) was stably expressed. Besides, AChE might be able to compensate the lack of BChE. Thus, further analyses using bone tissue specific AChE BChE cre-lox double knockout mice would be helpful.

  5. Effect of Salvia miltiorrhiza root extract on brain acetylcholinesterase and butyrylcholinesterase activities, their mRNA levels and memory evaluation in rats.

    PubMed

    Ozarowski, Marcin; Mikolajczak, Przemyslaw L; Piasecka, Anna; Kujawski, Radoslaw; Bartkowiak-Wieczorek, Joanna; Bogacz, Anna; Szulc, Michal; Kaminska, Ewa; Kujawska, Malgorzata; Gryszczynska, Agnieszka; Kachlicki, Piotr; Buchwald, Waldemar; Klejewski, Andrzej; Seremak-Mrozikiewicz, Agnieszka

    2017-05-01

    Salvia miltiorrhiza (Lamiaceae), one of the most important and popular plants of traditional medicine of Asia, is used for the prevention and treatment of cardiovascular diseases and in central nervous system disturbances. The main aim of this study was to assess the influence of subchronic (28-fold) administration of Salvia miltiorrhiza root extract (SE, 200mg/kg, p.o.) on behavioural activity and memory of rats and to evaluate the activities of cholinesterases (AChE and BuChE) and gene expression levels of AChE and BuChE as well as of beta-secretase (BACE1) in the hippocampus and frontal cortex in vivo. Huperzine A (HU, 0.5mg/kg b.w., p.o.) served as a positive control substance, whereas scopolamine (0.5mg/kg, i.p.) injection was used as a well-known model of memory impairment. The results showed that subchronic administration of SE led to an improvement of long-term memory of rats. Strong inhibition of AChE and BuChE mRNA transcription in the frontal cortex of rats treated with SE or HU was observed. The BACE1 transcript level was significantly decreased. AChE activity was statistically significantly inhibited in the frontal cortex and the hippocampus by SE (47% and 55%, respectively). Similar effects were observed in the case of HU. In summary, activity of SE provides evidence that the plant can be a source of drugs used in the treatment of Alzheimer disease.

  6. Synthesis and in vitro evaluation of bis-quaternary 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide derivatives as reactivators against sarin and VX inhibited human acetylcholinesterase (hAChE).

    PubMed

    Karade, Hitendra N; Valiveti, Aditya Kapil; Acharya, Jyotiranjan; Kaushik, Mahabir Parshad

    2014-05-01

    A series of bis-quaternary pyridinium derivatives 3a-3i of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide (2) have been synthesized. The synthesized pyridinium compounds have an amide group in conjugation to the oxime moiety. These compounds were evaluated in vitro for their reactivation efficacy against organophosphorus (OP) nerve agents (NAs) (sarin and VX) inhibited human erythrocyte ghost acetylcholinesterase (hAChE) and compared with the reactivation efficacy of 2-PAM and obidoxime. The pKa values of the synthesized compounds were found closer to the pKa values of 2- and 4-pyridinium oxime reactivators such as 2-PAM and obidoxime. Some of the compounds have shown better reactivation efficacy than 2-PAM, and obidoxime against sarin and VX inhibited AChE.

  7. Recent developments in the synthesis of acetylcholinesterase inhibitors.

    PubMed

    Marco, José L; Carreiras, M Carmo

    2003-09-01

    The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities of a series of pyrano[2,3-b]quinolines (2, 3), [1,8]naphthyridines (5, 6), 4-amino-2,3-diaryl-5,6,7,8-tetrahydrofuro[2,3-b]quinolines (11-13)/ 4-amino-6,7,8,9-tetrahydro-2,3-diphenyl-5H-cyclohepta[e]furo[2,3-b]pyridine (14), 4-amino-5,6,7,8-tetrahydro-2,3-diphenylthieno[2,3-b]quinoline (15)/ 4-amino-6,7,8,9-tetrahydro-2,3-diphenyl-5H-cyclohepta[e]thieno[2,3-b]pyridine (16) are described. These compounds are tacrine analogues that have been prepared from readily available polyfunctionalized ethyl [6-amino-5-cyano-4H-pyran]-3-carboxylates (9, 10), ethyl [6-amino-5-cyanopyridine]-3-carboxylates (7, 8), 2-amino-3-cyano-4,5-diarylfurans (17-19) and 2-amino-3-cyano-4,5-diphenylthiophene (20) via Friedländer condensation with selected ketones. These compounds are competitive and, in a few cases, non-competitive inhibitors for AChE, the most potent being compound (14), though three-fold less active than tacrine. The BuChE inhibitory activity is only significant in compounds 11 and 14, ten-fold less active than tacrine. Furthermore, the products 12 and 13 are selective and moderate AChE inhibitors.

  8. Blood and bronchoalveolar lavage fluid acetylcholinesterase levels following microinstillation inhalation exposure to sarin in Guinea pigs.

    PubMed

    Che, Magnus M; Conti, Michele; Boylan, Megan; Sciuto, Alfred M; Gordon, Richard K; Nambiar, Madhusoodana P

    2008-07-01

    We determined acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition in the bronchoalveolar lavage fluid (BALF) following inhalation exposure to chemical threat nerve agent (CTNA) sarin. Age- and weight-matched male guinea pigs were exposed to five different doses of sarin (169.3, 338.7, 508, 677.4, and 846.5 mg/m(3)) using a microinstillation inhalation exposure technique for 4 min. The technique involves aerosolization of the agent in the trachea using a microcatheter with a center hole that delivers the agent and multiple peripheral holes that pumps air to aerosolize the agent at the tip. Animals exposed to higher doses of sarin occasionally developed seizures and succumbed to death within 15 min after exposure. The LCt(50) for sarin using the microinstillation technique was determined to be close to 677.4 mg/m(3). Ear blood AChE activity showed a dose-dependent inhibition at 15 min postexposure. The inhibition of blood AChE remained constant over 35 and 55 min after sarin exposure indicating that there was no lung depot effect. Cardiac blood AChE and butyrylcholinesterase (BChE) activity in surviving animals euthanized at 24 h postexposure showed a dose-dependent inhibition with an inhibition of 60% at 677.4 and 846.5 mg/m(3) sarin exposure. AChE and BChE activity in bronchoalveolar lavage fluid (BALF) showed a slight increase at 338.7 to 677.4 mg/m(3) sarin exposure but a marginal inhibition at 169.3 mg/m(3). In contrast, the AChE protein levels determined by immunoblotting showed an increase at 169.3 mg/m(3) in the BALF. The BALF protein level, a biomarker of lung injury, was increased maximally at 338.7 mg/m(3) and that increase was dropped with an increase in the dose of sarin. The BALF protein levels correlated with the AChE and BChE activity. These data suggest that sarin microinstillation inhalation exposure results in respiratory toxicity and lung injury characterized by changes in lavage AChE, BChE, and protein levels.

  9. Fatty Acid Amide Hydrolase (FAAH), Acetylcholinesterase (AChE), and Butyrylcholinesterase (BuChE): Networked Targets for the Development of Carbamates as Potential Anti-Alzheimer's Disease Agents.

    PubMed

    Montanari, Serena; Scalvini, Laura; Bartolini, Manuela; Belluti, Federica; Gobbi, Silvia; Andrisano, Vincenza; Ligresti, Alessia; Di Marzo, Vincenzo; Rivara, Silvia; Mor, Marco; Bisi, Alessandra; Rampa, Angela

    2016-07-14

    The modulation of the endocannabinoid system is emerging as a viable avenue for the treatment of neurodegeneration, being involved in neuroprotective and anti-inflammatory processes. In particular, indirectly enhancing endocannabinoid signaling to therapeutic levels through FAAH inhibition might be beneficial for neurodegenerative disorders such as Alzheimer's disease, effectively preventing or slowing the progression of the disease. Hence, in the search for a more effective treatment for Alzheimer's disease, in this paper, the multitarget-directed ligand paradigm was applied to the design of carbamates able to simultaneously target the recently proposed endocannabinoid system and the classic cholinesterase system, and achieve effective dual FAAH/cholinesterase inhibitors. Among the two series of synthesized compounds, while some derivatives proved to be extremely potent on a single target, compounds 9 and 19 were identified as effective dual FAAH/ChE inhibitors, with well-balanced nanomolar activities. Thus, 9 and 19 might be considered as new promising candidates for Alzheimer's disease treatment.

  10. Measurement of p-nitrophenyl acetate esterase activity (EA), total antioxidant capacity (TAC), total oxidant status (TOS) and acetylcholinesterase (AChE) in gills and digestive gland of Mytilus galloprovincialis exposed to binary mixtures of Pb, Cd and Cu.

    PubMed

    Franco-Martinez, Lorena; Romero, Diego; García-Navarro, José A; Tecles, Fernando; Teles, Mariana; Tvarijonaviciute, Asta

    2016-12-01

    The aims of the present work were (1) to evaluate oxidative stress biomarkers and AChE in two tissues of wild mussel (Mytilus galloprovincialis) of high biochemical activity and accumulation capacity (gills and digestive gland) and (2) to study the behaviour of these biomarkers in presence of heavy metals. For this, EA, TOS, TAC and AChE were measured in tissues of mussels exposed to binary combination of Pb, Cd and Cu. Mussels (n = 36) were exposed to one of the binary mixtures of Pb (1000 μg L(-1)), Cd (100 μg L(-1)) and Cu (100 μg L(-1)) for 7 days, under controlled conditions. Gills and digestive gland were extracted and frozen at -80 °C until analysis. The automatic methods employed for the measurement of EA, TAC, TOS and AChE in M. galloprovincialis revealed higher levels of these biomarkers in digestive gland than gills. Study results suggest that gills would be the tissue of election for study oxidative stress markers, whereas digestive tissue should be selected for AChE measurements in case of evaluation of combined metal toxicity in mussels.

  11. Synthesis and in vitro kinetic evaluation of N-thiazolylacetamido monoquaternary pyridinium oximes as reactivators of sarin, O-ethylsarin and VX inhibited human acetylcholinesterase (hAChE).

    PubMed

    Valiveti, Aditya Kapil; Bhalerao, Uma M; Acharya, Jyotiranjan; Karade, Hitendra N; Acharya, Badri Narayan; Raviraju, G; Halve, Anand K; Kaushik, Mahabir Parshad

    2015-08-01

    Presently available medications for treatment of organiphosphorus poisoning are not sufficiently effective due to various pharmacological and toxicological reasons. In this regard, herein we report the synthesis of a series of N-thiazolylacetamide monoquaternary pyridinium oximes and its analogs (1a-1b to 6a-6b) with diversely substituted thiazole ring and evaluation of their in vitro reactivation efficacies against nerve agent (sarin, O-ethylsarin and VX) inhibited human erythrocyte acetylcholinesterase (hAChE). Reactivation kinetics was performed to determine dissociation constant (KD), reactivity rate constant (kr) and the second order rate constant (kr2) for all the compounds and compared their efficacies with commercial antidotes viz. 2-PAM and obidoxime. All the newly synthesized oximes were evaluated for their physicochemical parameters (pKa) and correlated with their respective reactivation efficacies to assess the capability of the oxime reactivator. Three of these novel compounds showed promising reactivation efficacies toward OP inhibited hAChE. Molecular docking studies were performed in order to correlate the reactivation efficacies with their interactions in the active site of the AChE.

  12. 1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies.

    PubMed

    Di Pietro, Ornella; Viayna, Elisabet; Vicente-García, Esther; Bartolini, Manuela; Ramón, Rosario; Juárez-Jiménez, Jordi; Clos, M Victòria; Pérez, Belén; Andrisano, Vincenza; Luque, F Javier; Lavilla, Rodolfo; Muñoz-Torrero, Diego

    2014-02-12

    A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (>11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

  13. Effect of five acetylcholinesterase reactivators on tabun-intoxicated rats: induction of oxidative stress versus reactivation efficacy.

    PubMed

    Pohanka, Miroslav; Karasova, Jana Zdarova; Musilek, Kamil; Kuca, Kamil; Kassa, Jiri

    2009-08-01

    Oxime reactivators HI-6, obidoxime, trimedoxime, K347 and K628 were investigated as drugs designed for treatment of tabun intoxication. The experiments were performed on rats in order to simulate real conditions. Rats were intoxicated with one LD(50 )of tabun and treated with atropine and mentioned reactivators. Activities of erythrocyte acetylcholinesterase (AChE), plasma butyrylcholinesterase (BChE) and brain AChE were measured as markers of reactivation efficacy. An estimation of low molecular weight antioxidant levels using cyclic voltammetry was the second examination parameter. The evaluation of cholinesterases activity showed good reactivation potency of blood AChE and plasma BChE by commercially available obidoxime and newly synthesized K347. The potency of oximes to reactivate brain AChE was lower due to the poor blood-brain barrier penetration of used compounds. Commercially available reactivator HI-6 and newly synthesized K628 caused oxidative stress measured by cyclic voltammetry as antioxidant level. The oxidative stress provoked by HI-6 and K628 was found to be significant on probability level P = 0.05. The others reactivators did not affect antioxidant levels.

  14. Acetylcholinesterase inhibitors: SAR and kinetic studies on omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxyaryl derivatives.

    PubMed

    Rampa, A; Piazzi, L; Belluti, F; Gobbi, S; Bisi, A; Bartolini, M; Andrisano, V; Cavrini, V; Cavalli, A; Recanatini, M; Valenti, P

    2001-11-08

    In this work, we further investigated a class of carbamic cholinesterase inhibitors introduced in a previous paper (Rampa et al. J. Med. Chem. 1998, 41, 3976). Some new omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxyaryl analogues were designed, synthesized, and evaluated for their inhibitory activity against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The structure of the lead compound (xanthostigmine) was systematically varied with the aim to optimize the different parts of the molecule. Moreover, such a structure-activity relationships (SAR) study was integrated with a kinetic analysis of the mechanism of AChE inhibition for two representative compounds. The structural modifications lead to a compound (12b) showing an IC(50) value for the AChE inhibition of 0.32 +/- 0.09 nM and to a group of BuChE inhibitors also active at the nanomolar level, the most potent of which (15d) was characterized by an IC(50) value of 3.3 +/- 0.4 nM. The kinetic analysis allowed for clarification of the role played by different molecular moieties with regard to the rate of AChE carbamoylation and the duration of inhibition. On the basis of the results presented here, it was concluded that the cholinesterase inhibitors of this class possess promising characteristics in view of a potential development as drugs for the treatment of Alzheimer's disease.

  15. Butyrylcholinesterase in guinea pig lung lavage: a novel biomarker to assess lung injury following inhalation exposure to nerve agent VX.

    PubMed

    Graham, Jacob R; Wright, Benjamin S; Rezk, Peter E; Gordon, Richard K; Sciuto, Alfred M; Nambiar, Madhusoodana P

    2006-06-01

    Respiratory disturbances play a central role in chemical warfare nerve agent (CWNA) induced toxicity; they are the starting point of mass casualty and the major cause of death. We developed a microinstillation technique of inhalation exposure to nerve agent VX and assessed lung injury by biochemical analysis of the bronchoalveolar lavage fluid (BALF). Here we demonstrate that normal guinea pig BALF has a significant amount of cholinesterase activity. Treatment with Huperzine A, a specific inhibitor of acetylcholinesterase (AChE), showed that a minor fraction of BALF cholinesterase is AChE. Furthermore, treatment with tetraisopropyl pyrophosphoramide (iso-OMPA), a specific inhibitor of butyrylcholinesterase (BChE), inhibited more than 90% of BChE activity, indicating the predominance of BChE in BALF. A predominance of BChE expression in the lung lavage was seen in both genders. Substrate specific inhibition indicated that nearly 30% of the cholinesterase in lung tissue homogenate is AChE. BALF and lung tissue AChE and BChE activities were strongly inhibited in guinea pigs exposed for 5 min to 70.4 and 90.4 microg/m3 VX and allowed to recover for 15 min. In contrast, BALF AChE activity was increased 63% and 128% and BChE activity was increased 77% and 88% after 24 h of recovery following 5 min inhalation exposure to 70.4 microg/m3 and 90.4 mg/m3 VX, respectively. The increase in BALF AChE and BChE activity was dose dependent. Since BChE is synthesized in the liver and present in the plasma, an increase in BALF indicates endothelial barrier injury and leakage of plasma into lung interstitium. Therefore, a measure of increased levels of AChE and BChE in the lung lavage can be used to determine the chronology of barrier damage as well as the extent of lung injury following exposure to chemical warfare nerve agents.

  16. Structural evidence that human acetylcholinesterase inhibited by tabun ages through O-dealkylation.

    PubMed

    Carletti, Eugénie; Colletier, Jacques-Philippe; Dupeux, Florine; Trovaslet, Marie; Masson, Patrick; Nachon, Florian

    2010-05-27

    Tabun is a warfare agent that inhibits human acetylcholinesterase (hAChE) by rapid phosphylation of the catalytic serine. A time-dependent reaction occurs on the tabun adduct, leading to an "aged" enzyme, resistant to oxime reactivators. The aging reaction may proceed via either dealkylation or deamidation, depending on the stereochemistry of the phosphoramidyl adduct. We solved the X-ray structure of aged tabun-hAChE complexed with fasciculin II, and we show that aging proceeds through O-dealkylation, in agreement with the aging mechanism that we determined for tabun-inhibited human butyrylcholinesterase and mouse acetylcholinesterase. Noteworthy, aging and binding of fasciculin II lead to an improved thermostability, resulting from additional stabilizing interactions between the two subdomains that face each other across the active site gorge. This first structure of hAChE inhibited by a nerve agent provides structural insight into the inhibition and aging mechanisms and a structural template for the design of molecules capable of reactivating aged hAChE.

  17. Acetylcholinesterase activity in the cerebrospinal fluid of dogs with seizures.

    PubMed

    Chai, Orit; Sommer, Adi; Zimmerman, Gabriel; Soreq, Hermona; Friedman, Alon; Bdolah-Abram, Tali; Aroch, Itamar; Shamir, Merav H

    2013-10-01

    Recent studies in animal models have focused on the role of cholinergic elements, mainly acetylcholinesterase (AChE) and the 'readthrough' acetylcholinesterase isoform (AChE-R), in seizures. A prospective double-masked study was conducted to assess the activity of AChE and AChE-R in cerebrospinal fluid (CSF) of 26 dogs post-seizure, 28 dogs with intervertebral disc disease (IVDD) and 16 healthy dogs. AChE was also measured in the serum in the post-seizure and IVDD groups. The results showed no significant differences in CSF AChE among the three groups. AChE-R was not detected in any dog and AChE in the serum was similar between groups. This preliminary study provides new information on AChE and AChE-R in the CSF and sera of dogs following naturally-occurring seizures.

  18. Electrophysiological and Ultrastructural Characterization of Neuromuscular Junctions in Diaphragm Muscle of Acetylcholinesterase Knockout Mice

    DTIC Science & Technology

    2008-04-01

    Electrophysiological and Ultrastructural Characterization of Neuromuscular Junctions in 5a. CONTRACT NUMBER Diaphragm Muscle of Acetylcholinesterase Knockout Mice...AChE +/+) and acetylcholinesterase knockout (AChE -/-) mice to determine the compensatory mechanism manifested by the neuromuscular junction to...had smaller nerve terminals and diminished pre- and postsynaptic surface contacts relative to neuromuscular junctions of AChE +/+ mice. The

  19. Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure.

    PubMed

    Brazzolotto, Xavier; Wandhammer, Marielle; Ronco, Cyril; Trovaslet, Marie; Jean, Ludovic; Lockridge, Oksana; Renard, Pierre-Yves; Nachon, Florian

    2012-08-01

    Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ≈ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 Å. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.

  20. Selective inhibition of human acetylcholinesterase by xanthine derivatives: in vitro inhibition and molecular modeling investigations.

    PubMed

    Mohamed, Tarek; Osman, Wesseem; Tin, Gary; Rao, Praveen P N

    2013-08-01

    The commonly used beverage and psychostimulant caffeine is known to inhibit human acetylcholinesterase enzyme. This pharmacological activity of caffeine is partly responsible for its cognition enhancing properties. However, the exact mechanisms of its binding to human cholinesterases (acetyl and butyrylcholinesterase; hAChE and hBuChE) are not well known. In this study, we investigated the cholinesterase inhibition by the xanthine derivatives caffeine, pentoxifylline, and propentofylline. Among them, propentofylline was the most potent AChE inhibitor (hAChE IC₅₀=6.40 μM). The hAChE inhibitory potency was of the order: caffeine (hAChE IC₅₀=7.25 μM)AChE IC₅₀=6.60 μM) ≤ propentofylline (hAChE IC₅₀=6.40 μM). These compounds were less potent relative to the reference agent donepezil (hAChE IC₅₀=0.04 μM). Moreover, they all exhibited selective inhibition of hAChE with no inhibition of hBuChE (IC₅₀>50 μM) relative to the reference agent donepezil (hBuChE IC₅₀=13.60 μM). Molecular modeling investigations indicate that caffeine binds primarily in the catalytic site (Ser203, Glu334 and His447) region of hAChE whereas pentoxifylline and propentofylline are able to bind to both the catalytic site and peripheral anionic site due to their increased bulk/size, thereby exhibiting superior AChE inhibition relative to caffeine. In contrast, their lack of hBuChE inhibition is due to a larger binding site and lack of key aromatic amino acids. In summary, our study has important implications in the development of novel caffeine derivatives as selective AChE inhibitors with potential application as cognitive enhancers and to treat various forms of dementia.

  1. Catalytic soman scavenging by Y337A/F338A acetylcholinesterase mutant assisted with novel site-directed aldoximes

    PubMed Central

    Kovarik, Zrinka; Hrvat, Nikolina Maček; Katalinić, Maja; Sit, Rakesh K.; Paradyse, Alexander; Žunec, Suzana; Musilek, Kamil; Fokin, Valery V.; Taylor, Palmer; Radić, Zoran

    2016-01-01

    Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin and paraoxon inhibition. We here demonstrate that ex vivo, in whole human blood, 1 μM soman was detoxified within 30 minutes when supplemented with 0.5 μM Y337A/F338A AChE and 100 μM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of forty-two pyridinium aldoximes, and five imidazole 2-aldoxime N-propyl pyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2–3 –fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack. PMID:25835984

  2. Genetic factors potentially reducing fitness cost of organophosphate-insensitive acetylcholinesterase(s) in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acaricidal activity of organophosphate (OP) and carbamate acaricides is believed to result from inhibition of acetylcholinesterase (AChE). Previous studies in Rhipicephalus (Boophilus) microplus demonstrated the presence of three presumptive AChE genes (BmAChEs). Biochemical characterization of re...

  3. Conjugates of γ-Carbolines and Phenothiazine as new selective inhibitors of butyrylcholinesterase and blockers of NMDA receptors for Alzheimer Disease.

    PubMed

    Makhaeva, Galina F; Lushchekina, Sofya V; Boltneva, Natalia P; Sokolov, Vladimir B; Grigoriev, Vladimir V; Serebryakova, Olga G; Vikhareva, Ekaterina A; Aksinenko, Alexey Yu; Barreto, George E; Aliev, Gjumrakch; Bachurin, Sergey O

    2015-08-18

    Alzheimer disease is a multifactorial pathology and the development of new multitarget neuroprotective drugs is promising and attractive. We synthesized a group of original compounds, which combine in one molecule γ-carboline fragment of dimebon and phenothiazine core of methylene blue (MB) linked by 1-oxo- and 2-hydroxypropylene spacers. Inhibitory activity of the conjugates toward acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and structurally close to them carboxylesterase (CaE), as well their binding to NMDA-receptors were evaluated in vitro and in silico. These newly synthesized compounds showed significantly higher inhibitory activity toward BChE with IC50 values in submicromolar and micromolar range and exhibited selective inhibitory action against BChE over AChE and CaE. Kinetic studies for the 9 most active compounds indicated that majority of them were mixed-type BChE inhibitors. The main specific protein-ligand interaction is π-π stacking of phenothiazine ring with indole group of Trp82. These compounds emerge as promising safe multitarget ligands for the further development of a therapeutic approach against aging-related neurodegenerative disorders such as Alzheimer and/or other pathological conditions.

  4. Mechanism-Based Analysis of Acetylcholinesterase Inhibitory Potency of Organophosphates, Carbamates, and Their Analogs

    EPA Science Inventory

    Acetylcholinesterase (AChE) is a key enzyme in the nervous system of animals, terminating impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Organophosphate (OP) and carbamate esters can inhibit acetylcholinesterase (AChE) by binding covalently to a s...

  5. AChE inhibition: one dominant factor for swimming behavior changes of Daphnia magna under DDVP exposure.

    PubMed

    Ren, Zongming; Zhang, Xu; Wang, Xiaoguang; Qi, Pingping; Zhang, Biao; Zeng, Yang; Fu, Rongshu; Miao, Mingsheng

    2015-02-01

    As a key enzyme that hydrolyzes the neurotransmitter acetylcholine in cholinergic synapses of both vertebrates and invertebrates, acetylcholinesterase (AChE) is strongly inhibited by organophosphates. AChE inhibition may induce the decrease of swimming ability. According to previous research, swimming behavior of different aquatic organisms could be affected by different chemicals, and there is a shortage of research on direct correlation analysis between swimming behavior and biochemical indicators. Therefore, swimming behavior and whole-body AChE activity of Daphnia magna under dichlorvos (DDVP) exposure were identified in order to clarify the relationship between behavioral responses and AChE inhibition in this study. In the beginning, AChE activity was similar in all treatments with the control. During all exposures, the tendency of AChE activity inhibition was the same as the behavioral responses of D. magna. The AChE activity of individuals without movement would decrease to about zero in several minutes. The correlation analysis between swimming behavior of D. magna and AChE activity showed that the stepwise behavioral response was mainly decided by AChE activity. All of these results suggested that the toxicity characteristics of DDVP as an inhibitor of AChE on the swimming behavior of organisms were the same, and the AChE activity inhibition could induce loss of the nerve conduction ability, causing hyperactivity, loss of coordination, convulsions, paralysis and other kinds of behavioral changes, which was illustrated by the stepwise behavioral responses under different environmental stresses.

  6. Introducing Dynamic Combinatorial Chemistry: Probing the Substrate Selectivity of Acetylcholinesterase

    ERIC Educational Resources Information Center

    Angelin, Marcus; Larsson, Rikard; Vongvilai, Pornrapee; Ramstrom, Olof

    2010-01-01

    In this laboratory experiment, college students are introduced to dynamic combinatorial chemistry (DCC) and apply it to determine the substrate selectivity of acetylcholinesterase (AChE). Initially, the students construct a chemical library of dynamically interchanging thioesters and thiols. Then, AChE is added and allowed to select and hydrolyze…

  7. Acetylcholinesterases of Blood-feeding Flies and Ticks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acetylcholinesterase (AChE) is the biochemical target of organophosphate (OP) and carbamate pesticides for invertebrates, vertebrate nerve agents, and AChE inhibitors used to reduce effects of Alzheimer’s disease. Organophosphate pesticides (OPs) are widely used to control blood-feeding arthropods, ...

  8. Butyrylcholinesterase for protection from organophosphorus poisons; catalytic complexities and hysteretic behavior

    PubMed Central

    Masson, Patrick; Lockridge, Oksana

    2009-01-01

    Butyrylcholinesterase is a promiscuous enzyme that displays complex kinetic behavior. It is toxicologically important because it detoxifies organophosphorus poisons (OP) by making a covalent bond with the OP. The OP and the butyrylcholinesterase are both inactivated in the process. Inactivation of butyrylcholinesterase has no adverse effects. However inactivation of acetylcholinesterase in nerve synapses can be lethal. OP-inhibited butyrylcholinesterase and acetylcholinesterase can be reactivated with oximes provided the OP has not aged. Strategies for preventing the toxicity of OP include a) treatment with an OP scavenger, b) reaction of nonaged enzyme with oximes, c) reactivation of aged enzyme, d) slowing down aging with peripheral site ligands, and e) design of mutants that rapidly hydrolyze OP. Option (a) has progressed through phase I clinical trials with human butyrylcholinesterase. Option (b) is in routine clinical use. The others are at the basic research level. Butyrylcholinesterase displays complex kinetic behavior including activation by positively charged esters, ability to hydrolyze amides, and a lag time (hysteresis) preceding hydrolysis of benzoylcholine and N-methyl indoxyl acetate. Mass spectrometry has identified new OP binding motifs on tyrosine and lysine in proteins that have no active site serine. It is proposed, but not yet proven, that low dose exposure involves OP modification of proteins that have no active site serine. PMID:20004171

  9. Effects of inescapable stress and treatment with pyridostigmine bromide on plasma butyrylcholinesterase and the acoustic startle response in rats.

    PubMed

    Servatius, R J; Ottenweller, J E; Guo, W; Beldowicz, D; Zhu, G; Natelson, B H

    2000-05-01

    Pyridostigmine bromide (PB) is a reversible, peripherally active inhibitor of acetylcholinesterase (AChE) activity, and is recommended by the military as a pretreatment against potential nerve gas exposure. Recent evidence suggests that exposure to inescapable stressors allows PB to cross the blood-brain barrier, and thereby affect central AChE activity in mice. Here, we evaluated the functional impact of a stress/PB treatment interaction on acoustic startle responding and plasma butyrylcholinesterase (BuChE) activity in male Sprague-Dawley rats. To model the treatment protocol used by the military, PB was delivered in the drinking water of rats for 7 consecutive days. The morning after the start of PB treatment, and for the next 6 days, half the rats were exposed to 1 h of supine restraint stress. We therefore employed a 2 x 2 (stress x PB treatment) between-groups design. Exposure to supine stress alone induced a persistent decrease in plasma BuChE activity. Further decreases in BuChE activity were not observed in rats exposed to supine restraint and PB treatment. Exposure to stress also induced an exaggerated startle response, evident on the last day of stress and 24 h after stressor cessation. Treatment with PB alone produced an exaggerated startle response over the same time period, albeit to a lesser degree. Although treatment with PB concurrent with stress did not produce further changes in either BuChE activity or acoustic startle responding, stress-induced alterations in drinking behavior (and thereby the dose of PB ingested) may have affected these results. Persistent stress-induced reductions in BuChE activity may increase the risk of adverse reactions to cholinomimetics.

  10. Muscle aches

    MedlinePlus

    ... common cause of muscle aches and pain is fibromyalgia , a condition that causes tenderness in your muscles ... imbalance, such as too little potassium or calcium Fibromyalgia Infections, including the flu, Lyme disease , malaria , muscle ...

  11. Design and synthesis of dual inhibitors of acetylcholinesterase and serotonin transporter targeting potential agents for Alzheimer's disease.

    PubMed

    Kogen, Hiroshi; Toda, Narihiro; Tago, Keiko; Marumoto, Shinji; Takami, Kazuko; Ori, Mayuko; Yamada, Naho; Koyama, Kazuo; Naruto, Shunji; Abe, Kazumi; Yamazaki, Reina; Hara, Takao; Aoyagi, Atsushi; Abe, Yasuyuki; Kaneko, Tsugio

    2002-10-03

    Highly efficient acetylcholinesterase (AChE) and serotonin transporter (SERT) dual inhibitors, (S)-4 and (R)-13 were designed and synthesized on the basis of the hypothetical model of AChE active site. Both compounds showed potent inhibitory activities against AChE and SERT. [structure: see text

  12. Safety of administration of human butyrylcholinesterase and its conjugates with soman or VX in rats.

    PubMed

    Genovese, Raymond F; Sun, Wei; Johnson, Christina C; Ditargiani, Robert C; Doctor, Bhupendra P; Saxena, Ashima

    2010-05-01

    We evaluated the effects of conjugated enzyme-nerve agent product resulting from the inhibition of bioscavenger human serum butyrylcholinesterase (Hu BChE) by nerve agents soman or VX. Rats were trained on a multiple Fixed-Ratio 32, Extinction 30 sec. (FR32, Ext30) schedule of food reinforcement and then injected (i.m.) with Hu BChE (30 mg/kg), equivalent amounts of Hu BChE-soman conjugate (GDC), Hu BChE-VX conjugate, oxotremorine (OXO) (0.316 mg/kg) or vehicle (n = 8, each group). On the day of injection and on 10 subsequent daily sessions, performance was evaluated on the FR32, Ext30 schedule. Neither conjugates nor Hu BChE produced a performance deficit under the schedule. OXO produced a substantial decrease in responding on the day of administration, with complete recovery observed on subsequent sessions. None of the treatments affected circulating acetylcholinesterase (AChE) activity when evaluated 24-72 hr after injection. The dose of Hu BChE produced a 20,000-fold increase above baseline in circulating BChE activity. Pathological evaluation of organ systems approximately 2 weeks following administration of conjugates or Hu BChE alone did not show toxicity. Taken together, these results suggest that Hu BChE - nerve agent conjugates produced following bioscavenger protection against nerve agents soman and VX do not appear to be particularly toxic. These results add to the safety assessment of Hu BChE as a bioscavenger countermeasure against nerve agent exposure.

  13. Overexpression of acetylcholinesterase gene in rice results in enhancement of shoot gravitropism.

    PubMed

    Yamamoto, Kosuke; Shida, Satoshi; Honda, Yoshihiro; Shono, Mariko; Miyake, Hiroshi; Oguri, Suguru; Sakamoto, Hikaru; Momonoki, Yoshie S

    2015-09-25

    Acetylcholine (ACh), a known neurotransmitter in animals and acetylcholinesterase (AChE) exists widely in plants, although its role in plant signal transduction is unclear. We previously reported AChE in Zea mays L. might be related to gravitropism based on pharmacological study using an AChE inhibitor. Here we clearly demonstrate plant AChE play an important role as a positive regulator in the gravity response of plants based on a genetic study. First, the gene encoding a second component of the ACh-mediated signal transduction system, AChE was cloned from rice, Oryza sativa L. ssp. Japonica cv. Nipponbare. The rice AChE shared high homology with maize, siratro and Salicornia AChEs. Similar to animal and other plant AChEs, the rice AChE hydrolyzed acetylthiocholine and propionylthiocholine, but not butyrylthiocholine. Thus, the rice AChE might be characterized as an AChE (E.C.3.1.1.7). Similar to maize and siratro AChEs, the rice AChE exhibited low sensitivity to the AChE inhibitor, neostigmine bromide, compared with the electric eel AChE. Next, the functionality of rice AChE was proved by overexpression in rice plants. The rice AChE was localized in extracellular spaces of rice plants. Further, the rice AChE mRNA and its activity were mainly detected during early developmental stages (2 d-10 d after sowing). Finally, by comparing AChE up-regulated plants with wild-type, we found that AChE overexpression causes an enhanced gravitropic response. This result clearly suggests that the function of the rice AChE relate to positive regulation of gravitropic response in rice seedlings.

  14. Natural AChE Inhibitors from Plants and their Contribution to Alzheimer’s Disease Therapy

    PubMed Central

    Murray, Ana Paula; Faraoni, María Belén; Castro, María Julia; Alza, Natalia Paola; Cavallaro, Valeria

    2013-01-01

    As acetylcholinesterase (AChE) inhibitors are an important therapeutic strategy in Alzheimer’s disease, efforts are being made in search of new molecules with anti-AChE activity. The fact that naturally-occurring compounds from plants are considered to be a potential source of new inhibitors has led to the discovery of an important number of secondary metabolites and plant extracts with the ability of inhibiting the enzyme AChE, which, according to the cholinergic hypothesis, increases the levels of the neurotransmitter acetylcholine in the brain, thus improving cholinergic functions in patients with Alzheimer’s disease and alleviating the symptoms of this neurological disorder. This review summarizes a total of 128 studies which correspond to the most relevant research work published during 2006-2012 (1st semester) on plant-derived compounds, plant extracts and essential oils found to elicit AChE inhibition. PMID:24381530

  15. Novel AChE Inhibitors for Sustainable Insecticide Resistance Management

    PubMed Central

    Alout, Haoues; Labbé, Pierrick; Berthomieu, Arnaud; Djogbénou, Luc; Leonetti, Jean-Paul; Fort, Philippe; Weill, Mylène

    2012-01-01

    Resistance to insecticides has become a critical issue in pest management and it is particularly chronic in the control of human disease vectors. The gravity of this situation is being exacerbated since there has not been a new insecticide class produced for over twenty years. Reasoned strategies have been developed to limit resistance spread but have proven difficult to implement in the field. Here we propose a new conceptual strategy based on inhibitors that preferentially target mosquitoes already resistant to a currently used insecticide. Application of such inhibitors in rotation with the insecticide against which resistance has been selected initially is expected to restore vector control efficacy and reduce the odds of neo-resistance. We validated this strategy by screening for inhibitors of the G119S mutated acetylcholinesterase-1 (AChE1), which mediates insensitivity to the widely used organophosphates (OP) and carbamates (CX) insecticides. PyrimidineTrione Furan-substituted (PTF) compounds came out as best hits, acting biochemically as reversible and competitive inhibitors of mosquito AChE1 and preferentially inhibiting the mutated form, insensitive to OP and CX. PTF application in bioassays preferentially killed OP-resistant Culex pipiens and Anopheles gambiae larvae as a consequence of AChE1 inhibition. Modeling the evolution of frequencies of wild type and OP-insensitive AChE1 alleles in PTF-treated populations using the selectivity parameters estimated from bioassays predicts a rapid rise in the wild type allele frequency. This study identifies the first compound class that preferentially targets OP-resistant mosquitoes, thus restoring OP-susceptibility, which validates a new prospect of sustainable insecticide resistance management. PMID:23056599

  16. Acute sarin exposure causes differential regulation of choline acetyltransferase, acetylcholinesterase, and acetylcholine receptors in the central nervous system of the rat.

    PubMed

    Khan, W A; Dechkovskaia, A M; Herrick, E A; Jones, K H; Abou-Donia, M B

    2000-09-01

    Acute neurotoxic effects of sarin (O:-isopropylmethylphosphonoflouridate) in male Sprague-Dawley rats were studied. The animals were treated with intramuscular (im) injections of either 1 x LD(50) (100 microg/kg), and sacrificed at 0. 5, 1, 3, 6, 15, or 20 h after treatment, or with im injections of either 0.01, 0.1, 0.5, or 1 x LD(50) and sacrificed 15 h after treatment. Plasma butyrylcholinesterase (BChE) and brain regional acetylcholinesterase (AChE) were inhibited (45-55%) by 30 min after the LD(50) dose. BChE in the plasma and AChE in cortex, brainstem, midbrain, and cerebellum remained inhibited for up to 20 h following a single LD(50) treatment. No inhibition in plasma BChE activity was observed 20 h after treatment with doses lower than the LD(50) dose. Midbrain and brainstem seem to be most responsive to sarin treatment at lower doses, as these regions exhibited inhibition (approximately 49% and 10%, respectively) in AChE activity following 0.1 x LD(50) treatment, after 20 h. Choline acetyltransferase (ChAT) activity was increased in cortex, brainstem, and midbrain 6 h after LD(50) treatment, and the elevated enzyme activity persisted up to 20 h after treatment. Cortex ChAT activity was significantly increased following a 0.1 x LD(50) dose, whereas brainstem and midbrain did not show any effect at lower doses. Treatment with an LD(50) dose caused a biphasic response in cortical nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (m2-mAChR) ligand binding, using [(3)H]cytisine and [(3)H]AFDX-384 as ligands for nAChR and mAChR, respectively. Decreases at 1 and 3 h and consistent increases at 6, 15, and 20 h in nAChR and m2-mAChR were observed following a single LD(50) dose. The increase in nAChR ligand binding densities was much more pronounced than in mAChR. These results suggest that a single exposure of sarin, ranging from 0.1 to 1 x LD(50), modulates the cholinergic pathways differently and thereby causes dysregulation in

  17. AChE and the amyloid precursor protein (APP) - Cross-talk in Alzheimer's disease.

    PubMed

    Nalivaeva, Natalia N; Turner, Anthony J

    2016-11-25

    The amyloid precursor protein (APP) and acetylcholinesterase (AChE) are multi-faceted proteins with a wide range of vital functions, both crucially linked with the pathogenesis of Alzheimer's disease (AD). APP is the precursor of the Aβ peptide, the pathological agent in AD, while AChE is linked to its pathogenesis either by increasing cholinergic deficit or exacerbating Aβ fibril formation and toxicity. As such, both proteins are the main targets in AD therapeutics with AChE inhibitors being currently the only clinically available AD drugs. In our studies we have demonstrated an important inter-relation in functioning of these proteins. Both can be released from the cell membrane and we have shown that AChE shedding involves a metalloproteinase-mediated mechanism which, like the α-secretase dependent cleavage of APP, is stimulated by cholinergic agonists. Overexpression of the neuronal specific isoform APP695 in neuronal cells substantially decreased levels of the AChE mRNA, protein and catalytic activity accompanied by a similar decrease in mRNA levels of the AChE membrane anchor, PRiMA (proline rich membrane anchor). We further established that this regulation does not involve APP processing and its intracellular domain (AICD) but requires the E1 region of APP, specifically its copper-binding domain. On the contrary, siRNA knock-down of APP in cholinergic SN56 cells resulted in a significant upregulation of AChE mRNA levels. Hence APP may influence AChE physiology while released AChE may regulate amyloidogenesis through multiple mechanisms suggesting novel therapeutic targets.

  18. Molecular Probe Analysis of Mammalian Brain Acetylcholinesterase

    DTIC Science & Technology

    1988-09-27

    Project and Degrees Awarded During this Reporting Period: Judith K. Marquis, Principal Investigator Thomas Biagioni , Senior Research Technician Robert...binding sites in nerve membrane vesicles. Comp. Biochem. Physiol. 80C: 203-205 (1985). 5. Volpe, L.S., T.M. Biagioni & J.K. Marquis: In vitro modulation of...Saxena, Vol. 6(1988Y.8 11. Marquis, J.K. & T.M. Biagioni : Selective inhibition of acetylcholinesterase and butyrylcholinesterase in human plasma

  19. Transcriptional regulation of acetylcholinesterase-associated collagen ColQ in fast- and slow-twitch muscle fibers.

    PubMed

    Ting, Annie K L; Siow, Nina L; Kong, L W; Tsim, Karl W K

    2005-12-15

    The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase (AChE) and butyrylcholinesterase at vertebrate neuromuscular junctions, which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal sequences, are encoded by transcripts ColQ-1 and ColQ-1a, which are differentially expressed in slow- and fast-twitch muscles in mammals, respectively. Both ColQ transcripts are derived from a single COLQ gene. Transcripts encoding ColQ increased during myogenic differentiation of C2C12 cells; the increase was in parallel with AChE catalytic subunit. Quantitative PCR analysis indicated that the increase during the myotube formation was due to the up regulation of ColQ-1 transcript instead of ColQ-1a. In order to reveal the regulatory mechanism of ColQ transcripts, two distinct promoters, pColQ-1 and pColQ-1a, were isolated from human COLQ gene. The ColQ promoters showed a muscle fiber type-specific expression pattern, and which was in line with the expression of endogenous transcript. After in vivo DNA transfection, pColQ-1 showed strong activity in slow-twitch muscle (e.g. soleus), while pColQ-1a was preferably expressed in fast-twitch muscle (e.g. tibialis). Mutation analysis of the ColQ promoters suggested that the muscle fiber type-specific expression pattern of ColQ transcripts was regulated by a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE). These results explain the specific expression patterns of collagen-tailed AChE in slow and fast muscle fibers.

  20. Morphogenic role for acetylcholinesterase in axonal outgrowth during neural development.

    PubMed Central

    Bigbee, J W; Sharma, K V; Gupta, J J; Dupree, J L

    1999-01-01

    Acetylcholinesterase (AChE) is the enzyme that hydrolyzes the neurotransmitter acetylcholine at cholinergic synapses and neuromuscular junctions. However, results from our laboratory and others indicate that AChE has an extrasynaptic, noncholinergic role during neural development. This article is a review of our findings demonstrating the morphogenic role of AChE, using a neuronal cell culture model. We also discuss how these data suggest that AChE has a cell adhesive function during neural development. These results could have additional significance as AChE is the target enzyme of agricultural organophosphate and carbamate pesticides as well as the commonly used household organophosphate chlorpyrifos (Dursban). Prenatal exposure to these agents could have adverse effects on neural development by interfering with the morphogenic function of AChE. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 PMID:10229710

  1. Acetylcholinesterase inhibitory properties of some benzoic acid derivatives

    NASA Astrophysics Data System (ADS)

    Yildiz, Melike; Kiliç, Deryanur; Ünver, Yaǧmur; Şentürk, Murat; Askin, Hakan; Küfrevioǧlu, Ömer Irfan

    2016-04-01

    Acetylcholinesterase (AChE) hydrolyses the neurotransmitter acetylcholine to acetic acid and choline. AChE inhibitors are used in treatment of several neurodegeneartive disorder and Alzheimer's disease. In the present study, inhibition of AChE with some benzoic acid derivatives were investigated. 3-Chloro-benzoic acid (1), 2-hydroxy-5-sulfobenzoic acid (2), 2-(sulfooxy) benzoic acid (3), 2-hydroxybenzoic acid (4), 2,3-dimethoxybenzoic (5), and 3,4,5-trimethoxybenzoic (6) were calculated IC50 values AChE enzyme. Kinetic investigations showed that similarly to AChE inhibitors. Benzoic acid derivatives (1-6) investigated are encouraging agents which may be used as lead molecules in order to derivative novel AChE inhibitors that might be useful in medical applications.

  2. Acetylcholinesterase inhibitory activity of uleine from Himatanthus lancifolius.

    PubMed

    Seidl, Cláudia; Correia, Beatriz L; Stinghen, Andréa E M; Santos, Cid A M

    2010-01-01

    Application of acetylcholinesterase (AChE) inhibitors is the primary treatment for Alzheimer's disease. Alkaloids, such as physostigmine, galanthamine, and huperzine A, play an important role as AChE inhibitors. The aim of this work was to evaluate Himatanthus lancifolius (Muell. Arg.) Woodson, a Brazilian species of Apocynaceae, and its main indole alkaloid uleine, in order to identify new AChE inhibitors. The plant fluid extract, fractions, and uleine were tested for AChE inhibitory activity using Ellman's colorimetric method for thin-layer chromatography (TLC), 96-well microplates, and also Marston's TLC colorimetric method. Both TLC assays showed similar results. At 5 mg/mL, the fluid extract inhibited the AChE enzyme by (50.71 +/- 8.2)%. The ethyl acetate fraction exhibited the highest level of AChE inhibition, followed by the dichloromethane fraction. The isolated alkaloid uleine displayed an IC50 value of 0.45 microM.

  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. Structure-activity relationship for the reactivators of acetylcholinesterase inhibited by nerve agent VX.

    PubMed

    Kuca, Kamil; Musilek, Kamil; Jun, Daniel; Karasova, Jana; Soukup, Ondrej; Pejchal, Jaroslav; Hrabinova, Martina

    2013-08-01

    Nerve agents such as sarin, VX and tabun are organophosphorus compounds able to inhibit an enzyme acetylcholinesterase (AChE). AChE reactivators and anticholinergics are generally used as antidotes in the case of intoxication with these agents. None from the known AChE reactivators is able to reactivate AChE inhibited by all kinds of nerve agents. In this work, reactivation potency of seventeen structurally different AChE reactivators was tested in vitro and subsequently, relationship between their chemical structure and biological activity was outlined. VX was chosen as appropriate member of the nerve agent family.

  5. An acetylcholinesterase-inspired biomimetic toxicity sensor.

    PubMed

    Wujcik, Evan K; Londoño, Nicolas J; Duirk, Stephen E; Monty, Chelsea N; Masel, Richard I

    2013-05-01

    This work demonstrates the ability of an acetylcholinesterase-inspired biomimetic sensor to accurately predict the toxicity of acetylcholinesterase (AChE) inhibitors. In surface waters used for municipal drinking water supplies, numerous pesticides and other anthropogenic chemicals have been found that inhibit AChE; however, there is currently no portable toxicity assay capable of determining the potential neurotoxicity of water samples and complex mixtures. Biological assays have been developed to determine the toxicity of unknown samples, but the short shelf-life of cells and other biological materials often make them undesirable for use in portable assays. Chemical methods and structure-activity-relationships, on the other hand, require prior knowledge on the compounds of interest that is often unavailable when analyzing environmental samples. In the toxicity assay presented here, the acetylcholinesterase enzyme has been replaced with 1-phenyl-1,2,3-butanetrione 2-oxime (PBO) a biomimetic compound that is structurally similar to the AChE active site. Using a biomimetic compound in place of the native enzyme allows for a longer shelf-life while maintaining the selective and kinetic ability of the enzyme itself. Previous work has shown the success of oxime-based sensors in the selective detection of AChE inhibitors and this work highlights the ability of an AChE-inspired biomimetic sensor to accurately predict the toxicity (LD50 and LC50) for a range of AChE inhibitors. The biomimetic assay shows strong linear correlations to LD50 (oral, rat) and LC50 (fish) values. Using a test set of eight AChE inhibitors, the biomimetic assay accurately predicted the LC50 value for 75% of the inhibitors within one order of magnitude.

  6. Analysis of AchE and LDH in mollusc, Lamellidens marginalis after exposure to chlorpyrifos.

    PubMed

    Amanullah, B; Stalin, A; Prabu, P; Dhanapal, S

    2010-07-01

    The enzymes Acetylcholinesterase (AchE) and Lactatedehydrogenase (LDH) are used as biological markers in the present study. Enzymes are highly sensitive and used to evaluate the biological effects of organophosphate pesticide chlorpyrifos in freshwater mussel Lamellidens marginalis. The test organisms were exposed to sub-lethal concentration (5 ppm) of chlorpyrifos for 30 days and allowed to recover for seven days. A distinct reduction of the enzyme AchE (34 +/- 3.3 U l(-1)) was found in the treated hepatopancreas. A significant increase in LDH activity in gill, hepatopancreas and muscle was observed. There was a significant recovery in AchE and LDH in the different tissues, after seven days recovery period.. Hence, the changes in the enzymes are found as the best biomarkering tool to evaluate the effect of organophosphate pesticide chlorpyrifos on the aquatic biota.

  7. Maize acetylcholinesterase is a positive regulator of heat tolerance in plants.

    PubMed

    Yamamoto, Kosuke; Sakamoto, Hikaru; Momonoki, Yoshie S

    2011-11-01

    We previously reported that native tropical zone plants showed high acetylcholinesterase (AChE) activity during heat stress, and that AChE activity in endodermal cells of maize seedlings was increased by heat treatment. However, the physiological role of AChE in heat stressed plants is still unclear. Here we report (1) tissue-specific expression and subcellular localization of maize AChE, (2) elevation of AChE activity and possible post-translational modifications of this enzyme under heat stress, and (3) involvement of AChE in plant heat stress tolerance. Maize AChE was mainly expressed in coleoptile nodes and seeds. Maize AChE fused with green fluorescent protein (GFP) was localized in extracellular spaces of transgenic rice plants. Therefore, in maize coleoptile nodes and seeds AChE mainly functions in the cell wall matrix. After heat treatment, enhanced maize AChE activity was observed by in vitro activity measurement and by in situ cytochemical staining; transcript and protein levels, however, were not changed. Protein gel blot analysis revealed two AChE isoforms (upper and lower); the upper-form gradually disappeared after heat treatment. Thus, maize AChE activity might be enhanced through a post-translational modification response to heat stress. Finally, we found that overexpression of maize AChE in transgenic tobacco plants enhanced heat tolerance relative to that of non-transgenic plants, suggesting AChE plays a positive role in maize heat tolerance.

  8. Interactions of AChE with Aβ Aggregates in Alzheimer's Brain: Therapeutic Relevance of IDN 5706.

    PubMed

    Carvajal, Francisco J; Inestrosa, Nibaldo C

    2011-01-01

    Acetylcholinesterase (AChE; EC 3.1.1.7) plays a crucial role in the rapid hydrolysis of the neurotransmitter acetylcholine, in the central and peripheral nervous system and might also participate in non-cholinergic mechanism related to neurodegenerative diseases. Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, amyloid-β (Aβ) peptide accumulation and synaptic alterations. We have previously shown that AChE is able to accelerate the Aβ peptide assembly into Alzheimer-type aggregates increasing its neurotoxicity. Furthermore, AChE activity is altered in brain and blood of Alzheimer's patients. The enzyme associated to amyloid plaques changes its enzymatic and pharmacological properties, as well as, increases its resistant to low pH, inhibitors and excess of substrate. Here, we reviewed the effects of IDN 5706, a hyperforin derivative that has potential preventive effects on the development of AD. Our results show that treatment with IDN 5706 for 10 weeks increases brain AChE activity in 7-month-old double transgenic mice (APP(SWE)-PS1) and decreases the content of AChE associated with different types of amyloid plaques in this Alzheimer's model. We concluded that early treatment with IDN 5706 decreases AChE-Aβ interaction and this effect might be of therapeutic interest in the treatment of AD.

  9. Butyrylcholinesterase as a Blood Biomarker in Neuroblastoma.

    PubMed

    Coulter, Don W; Boettner, Angela D; Kortylewicz, Zbigniew P; Enke, Stephen P; Luther, Jake A; Verma, Vivek; Baranowska-Kortylewicz, Janina

    2017-04-03

    Blood-based biomarkers are important in the detection of the disease and in the assessment of responses to therapy. In this study, butyrylcholinesterase was evaluated as a potential biomarker in newly diagnosed neuroblastoma (NB) patients at diagnosis and longitudinally during treatment. Plasma butyrylcholinesterase activities in age-matched and sex-matched children were used as controls. Pretreatment butyrylcholinesterase levels in NB subjects are on an average 2 times lower than butyrylcholinesterase levels in healthy subjects. Significantly, butyrylcholinesterase activities are ∼40% lower in MYCN-amplified as compared with nonamplified disease. As the course of chemotherapy progresses, butyrylcholinesterase activities recover and normalize to control values. The evident response to treatment indicates that plasma butyrylcholinesterase is a good biomarker of tumor response to therapy. Depressed butyrylcholinesterase levels in NB subjects are not caused by hepatic deficits suggesting a specific role for butyrylcholinesterase in NB. Further examination of the mechanism of altered butyrylcholinesterase production require an animal model that best approximates human condition. Studies in mice show that murine NB allografts significantly reduce butyrylcholinesterase activity in plasma. This finding correlates with changes observed in NB patients. In contrast, human NB xenografts produce the opposite effect, that is, butyrylcholinesterase plasma levels rise as the xenograft size increases. In the absence of any liver damage, dissimilarities between butyrylcholinesterase production in murine and human NB models suggest species-specific signaling pathways. This disparity also suggests that human NB xenograft mouse models do not approximate the human disease.

  10. Acetylcholinesterase and Nissl staining in the same histological section.

    PubMed

    Shipley, M T; Ennis, M; Behbehani, M M

    1989-12-18

    Acetylcholinesterase (AChE) enzyme histochemistry and Nissl staining are commonly utilized in neural architectonic studies. However, the opaque reaction deposit produced by the most commonly used AChE histochemical methods is not compatible with satisfactory Nissl staining. As a result, precise correlation of AChE and Nissl staining necessitates time-consuming comparisons of adjacent sections which may have differential shrinkage. Here, we have modified the Koelle-Friedenwald histochemical reaction for AChE by omitting the final intensification steps. The modified reaction yields a non-opaque reaction product that is selectively visualized by darkfield illumination. This non-intensified darkfield AChE (NIDA) reaction allows clear visualization of Nissl staining in the same histological section. This combined AChE-Nissl method greatly facilitates detailed correlation of enzyme and cytoarchitectonic organization.

  11. Crystal structure of acetylcholinesterase catalytic subunits of the malaria vector Anopheles gambiae.

    PubMed

    Han, Qian; Wong, Dawn M; Robinson, Howard; Ding, Haizhen; Lam, Polo C H; Totrov, Maxim M; Carlier, Paul R; Li, Jianyong

    2017-03-01

    Acetylcholinesterase (AChE) hydrolyzes the neurotransmitter acetylcholine at cholinergic synapses in the central nervous system (Toutant, 1989). Inhibition of the enzyme in insects could lead to the death of insects rapidly; thus AChE has been a molecular target for developing insecticides. This article is protected by copyright. All rights reserved.

  12. Cholinesterases in development: AChE as a firewall to inhibit cell proliferation and support differentiation.

    PubMed

    Layer, Paul G; Klaczinski, Janine; Salfelder, Anika; Sperling, Laura E; Thangaraj, Gopenath; Tuschl, Corina; Vogel-Höpker, Astrid

    2013-03-25

    Acetylcholinesterase (AChE) is a most remarkable protein, not only because it is one of the fastest enzymes in nature, but also since it appears in many molecular forms and is regulated by elaborate genetic networks. AChE is expressed in many tissues during development and in mature organisms, as well as in healthy and diseased states. In search for alternative, "non-classical" functions of cholinesterases (ChEs), AChE could either work within the frame of classic cholinergic systems, but in non-neural tissues ("non-synaptic function"), or act non-enzymatically. Here, we review briefly some of the major ideas and advances of this field, and report on some recent progress from our own experimental work, e.g. that (i) non-neural ChEs have pronounced, predominantly enzymatic effects on early embryonic (limb) development in chick and mouse, that (ii) retinal R28 cells of the rat overexpressing synaptic AChE present a significantly decreased cell proliferation, and that (iii) in developing chick retina ACh-synthesizing and ACh-degrading cells originate from the same postmitotic precursor cells, which later form two locally opposing cell populations. We suggest that such distinct distributions of ChAT(+) vs. AChE(+) cells in the inner half retina provide graded distributions of ACh, which can direct cell differentiation and network formation. Thus, as corroborated by works from many labs, AChE can be considered a highly co-opting protein, which can combine enzymatic and non-enzymatic functions within one molecule.

  13. Acetylcholinesterase modulates neurite outgrowth on fibronectin.

    PubMed

    Giordano, C; Poiana, G; Augusti-Tocco, G; Biagioni, S

    2007-05-04

    Acetylcholinesterase (AChE) has been reported to be involved in the modulation of neurite outgrowth. To understand the role played by different domains, we transfected neuroblastoma cells with three constructs containing the invariant region of AChE, differing in the exon encoding the C-terminus and therefore in AChE cellular fate and localization. All isoforms increased neurite extension, suggesting the involvement of the invariant domain [A. De Jaco, G. Augusti-Tocco, S. Biagioni, Alternative AChE molecular forms exhibit similar ability to induce neurite outgrowth, J. Neurosci. Res. 70 (2002) 756-765]. The peripheral anionic site (PAS) is encoded by invariant exons and represents the domain involved in non-cholinergic functions of AChE. Masking of PAS with fasciculin results in a significant decrease of neurite outgrowth in all clones overexpressing AChE. A strong reduction was also observed when clones were cultured on fibronectin. Treatment of clones with fasciculin, therefore masking PAS, abolished the fibronectin-induced reduction. The inhibition of the catalytic site cannot revert the fibronectin effect. Finally, when clones were cultured on fibronectin in the presence of heparin, a ligand of fibronectin, the inhibitory effect was completely reversed. Our results indicate that PAS could directly or indirectly mediate AChE/fibronectin interactions.

  14. A novel role for synaptic acetylcholinesterase as an apoptotic deoxyribonuclease

    PubMed Central

    Du, Aiying; Xie, Jing; Guo, Kaijie; Yang, Lei; Wan, Yihan; OuYang, Qi; Zhang, Xuejin; Niu, Xin; Lu, Lu; Wu, Jun; Zhang, Xuejun

    2015-01-01

    In addition to terminating neurotransmission by hydrolyzing acetylcholine, synaptic acetylcholinesterase (AChES) has been found to have a pro-apoptotic role. However, the underlying mechanism has rarely been investigated. Here, we report a nuclear translocation-dependent role for AChES as an apoptotic deoxyribonuclease (DNase). AChES polypeptide binds to and cleaves naked DNA at physiological pH in a Ca2+–Mg2+-dependent manner. It also cleaves chromosomal DNA both in pre-fixed and in apoptotic cells. In the presence of a pan-caspase inhibitor, the cleavage still occurred after nuclear translocation of AChES, implying that AChES-DNase acts in a CAD- and EndoG-independent manner. AChE gene knockout impairs apoptotic DNA cleavage; this impairment is rescued by overexpression of the wild-type but not (aa 32–138)-deleted AChES. Furthermore, in comparison with the nuclear-localized wild-type AChES, (aa 32–138)-deleted AChES loses the capacity to initiate apoptosis. These observations confirm that AChES mediates apoptosis via its DNase activity. PMID:27462404

  15. Brain acetylcholinesterase of jaguar cichlid (Parachromis managuensis): From physicochemical and kinetic properties to its potential as biomarker of pesticides and metal ions.

    PubMed

    Araújo, Marlyete Chagas de; Assis, Caio Rodrigo Dias; Silva, Luciano Clemente; Machado, Dijanah Cota; Silva, Kaline Catiely Campos; Lima, Ana Vitória Araújo; Carvalho, Luiz Bezerra; Bezerra, Ranilson de Souza; Oliveira, Maria Betânia Melo de

    2016-08-01

    This contribution aimed to characterize physicochemical and kinetic parameters of the brain cholinesterases (ChEs) from Parachromis managuensis and investigate the in vitro effects of pesticides and metal ions on its activity intending to propose as biomarker. This species is suitable for this investigation because (1) it was recently introduced in Brazil becoming invasive (no restrictions on capture) and (2) occupies the top of the food chain (being subject to bioaccumulation). The enzyme extract was exposed to 10 metal ions (Al(3+), Ba(2+), Cd(2+), Cu(2+), Hg(2+), Mg(2+), Mn(2+), Pb(2+), Fe(2+) and Zn(2+)) and ChEs selective inhibitors (BW284c51, Iso-OMPA, neostigmine and serine). The extract was also incubated with organophosphate (dichlorvos) and carbamate pesticides (carbaryl and carbofuran). Inhibition parameters (IC20, IC50 and ki) were determined. Selective inhibitors and kinetic parameters confirmed acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) -like as responsible for the ChE activities, most AChE. The IC50 values for pesticides were: 1.68μM (dichlorvos); 4.35μM (carbaryl) and 0.28μM (carbofuran). Most of the analyzed ions did not show significant effect at 1mM (p=0.05), whereas the following ions inhibited the enzyme activity in the order: Hg(2+)>Cu(2+)>Cd(2+)>Zn(2+). Mercury ion strongly inhibited the enzyme activity (IC20=0.7μM). The results about allow to conclude that P. managuensis brain AChE is a potential biomarker for heavy metals and pesticides under study, mainly for the carbamate carbofuran once it was capable to detect 6-fold lower levels than the limit concentration internationally recommended.

  16. Synthesis and in vitro reactivation study of isonicotinamide derivatives of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide as reactivators of Sarin and VX inhibited human acetylcholinesterase (hAChE).

    PubMed

    Karade, Hitendra N; Raviraju, G; Acharya, B N; Valiveti, Aditya Kapil; Bhalerao, Uma; Acharya, Jyotiranjan

    2016-09-15

    Previously (Karade et al., 2014), we have reported the synthesis and in vitro evaluation of bis-pyridinium derivatives of pyridine-3-yl-(2-hydroxyimino acetamide), as reactivators of sarin and VX inhibited hAChE. Few of the molecules showed superior in vivo protection efficacy (mice model) (Kumar et al., 2014; Swami et al., 2016) in comparison to 2-PAM against DFP and sarin poisoning. Encouraged by these results, herein we report the synthesis and in vitro evaluation of isonicotinamide derivatives of pyridine-3-yl-(2-hydroxyimino acetamide) (4a-4d) against sarin and VX inhibited erythrocyte ghost hAChE. Reactivation kinetics of these compounds was studied and the determined kinetic parameters were compared with that of commercial reactivators viz. 2-PAM and obidoxime. In comparison to 2-PAM and obidoxime, oxime 4a and 4b exhibited enhanced reactivation efficacy toward sarin inhibited hAChE while oxime 4c showed far greater reactivation efficacy toward VX inhibited hAChE. The acid dissociation constant and IC50 values of these oximes were determined and correlated with the observed reactivation potential.

  17. Carrageenans solubilize asymmetric acetylcholinesterase from nicotinic cholinergic synapses.

    PubMed

    von Bernhardi, R; Ayal, H; Inestrosa, N C

    1990-01-01

    1. Acetylcholinesterase (AChE) catalyzes the hydrolysis of acetylcholine at cholinergic synapses in both vertebrate and invertebrates organisms. 2. The asymmetric synaptic AChE is attached to the extracellular matrix (ECM) of the neuromuscular junction through heparin sulphate proteoglycans (HSPGs). 3. It has been shown previously that heparin-like glycosaminoglycans (GAGs) can solubilize this enzyme from the cholinergic synapses. 4. The present paper describes the solubilization of asymmetric AChE by different marine macroalgal polysaccharides, called carrageenans. 5. Important differences were found among all the carrageenans tested; they released 15-50% of the total AChE activity normally solubilized by heparin. 6. Carrageenans extracted from tetrasporic stages of Iridaea ciliata and I. membranacea were always better extracting agents than those from the cystocarpic stages of these algae, suggesting that lambda-like carrageenans are involved. 7. This hypothesis was confirmed by extracting AChE with purified carrageenans.

  18. Molecular Characterization of Maize Acetylcholinesterase. A Novel Enzyme Family in the Plant Kingdom1

    PubMed Central

    Sagane, Yoshimasa; Nakagawa, Tomoyuki; Yamamoto, Kosuke; Michikawa, Soichi; Oguri, Suguru; Momonoki, Yoshie S.

    2005-01-01

    Acetylcholinesterase (AChE) has been increasingly recognized in plants by indirect evidence of its activity. Here, we report purification and cloning of AChE from maize (Zea mays), thus providing to our knowledge the first direct evidence of the AChE molecule in plants. AChE was identified as a mixture of disulfide- and noncovalently linked 88-kD homodimers consisting of 42- to 44-kD polypeptides. The AChE hydrolyzed acetylthiocholine and propyonylthiocholine, but not S-butyrylthiocholine, and the AChE-specific inhibitor neostigmine bromide competitively inhibited its activity, implying that maize AChE functions in a similar manner as the animal enzyme. However, kinetic analyses indicated that maize AChE showed a lower affinity to substrates and inhibitors than animal AChE. The full-length cDNA of maize AChE gene is 1,471 nucleotides, which encode a protein having 394 residues, including a signal peptide. The deduced amino acid sequence exhibited no apparent similarity with that of the animal enzyme, although the catalytic triad was the same as in the animal AChE. In silico screening indicated that maize AChE homologs are widely distributed in plants but not in animals. These findings lead us to propose that the AChE family, as found here, comprises a novel family of the enzymes that is specifically distributed in the plant kingdom. PMID:15980188

  19. Acetylcholinesterase of the Sand Fly, Phlebotomus papatasi (Scopoli): cDNA Sequence, Baculovirus Expression, and Biochemical Properties

    DTIC Science & Technology

    2013-01-01

    papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of...identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17...classified as specific for butyrylcholinesterase. The enzyme is strongly inhibited by paraoxon and malaoxon (oxidized forms of parathion and malathion ) and

  20. New Acetylcholinesterase Inhibitors for Alzheimer's Disease

    PubMed Central

    Mehta, Mona; Adem, Abdu; Sabbagh, Marwan

    2012-01-01

    Acetylcholinesterase (AChE) remains a highly viable target for the symptomatic improvement in Alzheimer's disease (AD) because cholinergic deficit is a consistent and early finding in AD. The treatment approach of inhibiting peripheral AchE for myasthenia gravis had effectively proven that AchE inhibition was a reachable therapeutic target. Subsequently tacrine, donepezil, rivastigmine, and galantamine were developed and approved for the symptomatic treatment of AD. Since then, multiple cholinesterase inhibitors (ChEI) continue to be developed. These include newer ChEIs, naturally derived ChEIs, hybrids, and synthetic analogues. In this paper, we summarize the different types of ChEIs in development and their respective mechanisms of actions. This pharmacological approach continues to be active with many promising compounds. PMID:22216416

  1. Acetylcholinesterases of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene identification, expression and biochemical properties of recombinant proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhipicephalus (Boophilus) microplus (Bm) is a vector of bovine babesiosis and anaplasmosis. Tick resistance to organophosphate (OP) acaricide involves acetylcholinesterase (AChE) insensitivity to OP and metabolic detoxification. In vitro expression of Bm genes encoding AChE allowed biochemical chara...

  2. Acetylcholinesterase of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene identification, expression, and biochemical properties of recombinant proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhipicephalus (Boophilus) microplus (Bm) ticks are vectors of bovine babesiosis and anaplasmosis. Tick resistance to organophosphate (OP) acaricide involves acetylcholinesterase (AChE) insensitivity to OP and metabolic detoxification. Sequencing and in vitro expression of Bm genes encoding AChE allo...

  3. Complexity of acetylcholinesterases in biting flies and ticks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acetylcholinesterase (AChE) inhibitors function as pesticides for invertebrates, vertebrate nerve agents, and medicine to reduce cognitive effects of Alzheimer’s disease. Organophosphate (OP) pesticides have been widely used to control biting flies and ticks, however, OP-resistance has compromised c...

  4. Are soluble and membrane-bound rat brain acetylcholinesterase different

    SciTech Connect

    Andres, C.; el Mourabit, M.; Stutz, C.; Mark, J.; Waksman, A. )

    1990-11-01

    Salt-soluble and detergent-soluble acetylcholinesterases (AChE) from adult rat brain were purified to homogeneity and studied with the aim to establish the differences existing between these two forms. It was found that the enzymatic activities of the purified salt-soluble AChE as well as the detergent-soluble AChE were dependent on the Triton X-100 concentration. Moreover, the interaction of salt-soluble AChE with liposomes suggests amphiphilic behaviour of this enzyme. Serum cholinesterase (ChE) did not bind to liposomes but its activity was also detergent-dependent. Detergent-soluble AChE remained in solution below critical micellar concentrations of Triton X-100. SDS polyacrylamide gel electrophoresis of purified, Biobeads-treated and iodinated detergent-soluble 11 S AChE showed, under non reducing conditions, bands of 69 kD, 130 kD and greater than 250 kD corresponding, respectively, to monomers, dimers and probably tetramers of the same polypeptide chain. Under reducing conditions, only a 69 kD band was detected. It is proposed that an amphiphilic environment stabilizes the salt-soluble forms of AChE in the brain in vivo and that detergent-soluble Biobeads-treated 11 S AChE possess hydrophobic domain(s) different from the 20 kD peptide already described.

  5. Active Acetylcholinesterase Immobilization on a Functionalized Silicon Surface.

    PubMed

    Khaldi, K; Sam, S; Gouget-Laemmel, A C; Henry de Villeneuve, C; Moraillon, A; Ozanam, F; Yang, J; Kermad, A; Ghellai, N; Gabouze, N

    2015-08-04

    In this work, we studied the attachment of active acetylcholinesterase (AChE) enzyme on a silicon substrate as a potential biomarker for the detection of organophosphorous (OP) pesticides. A multistep functionalization strategy was developed on a crystalline silicon surface: a carboxylic acid-terminated monolayer was grafted onto a hydrogen-terminated silicon surface by photochemical hydrosilylation, and then AChE was covalently attached through amide bonds using an activation EDC/NHS process. Each step of the modification was quantitatively characterized by ex-situ Fourier transform infrared spectroscopy in attenuated-total-reflection geometry (ATR-FTIR) and atomic force microscopy (AFM). The kinetics of enzyme immobilization was investigated using in situ real-time infrared spectroscopy. The enzymatic activity of immobilized acetylcholinesterase enzymes was determined with a colorimetric test. The surface concentration of active AChE was estimated to be Γ = 1.72 × 10(10) cm(-2).

  6. Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1

    PubMed Central

    Sperling, Laura E.; Klaczinski, Janine; Schütz, Corina; Rudolph, Lydia; Layer, Paul G.

    2012-01-01

    The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1. PMID:22570738

  7. Plant-parasitic Nematode Acetylcholinesterase Inhibition by Carbamate and Organophosphate Nematicides.

    PubMed

    Opperman, C H; Chang, S

    1990-10-01

    The sensitivity of acetylcholinesterases (ACHE) isolated from the plant-parasitic nematodes Meloidogyne arenaria, M. incognita, and Heterodera glycines and the free-living nematode Caenorhabditis elegans to carbamate and organophosphate nematicides was examined. The AChE from plant-parasitic nematode species were more sensitive to carbamate inhibitors than was AChE from C. elegans, but response to the organophosphates was approximately equivalent. The sulfur-containing phosphate nematicides were poor inhibitors of nematode acetylcholinesterase, but treatment with an oxidizing agent greatly improved inhibition. Behavioral bioassays with living nematodes revealed a poor relationship between enzyme inhibition and expression of symptoms in live nematodes.

  8. Interaction of Acetylcholinesterase with Neurexin-1β regulates Glutamatergic Synaptic stability in Hippocampal neurons

    PubMed Central

    2014-01-01

    Background Excess expression of acetylcholinesterase (AChE) in the cortex and hippocampus causes a decrease in the number of glutamatergic synapses and alters the expression of neurexin and neuroligin, trans-synaptic proteins that control synaptic stability. The molecular sequence and three-dimensional structure of AChE are homologous to the corresponding aspects of the ectodomain of neuroligin. This study investigated whether excess AChE interacts physically with neurexin to destabilize glutamatergic synapses. Results The results showed that AChE clusters colocalized with neurexin assemblies in the neurites of hippocampal neurons and that AChE co-immunoprecipitated with neurexin from the lysate of these neurons. Moreover, when expressed in human embryonic kidney 293 cells, N-glycosylated AChE co-immunoprecipitated with non-O–glycosylated neurexin-1β, with N-glycosylation of the AChE being required for this co-precipitation to occur. Increasing extracellular AChE decreased the association of neurexin with neuroligin and inhibited neuroligin-induced synaptogenesis. The number and activity of excitatory synapses in cultured hippocampal neurons were reduced by extracellular catalytically inactive AChE. Conclusions Excessive glycosylated AChE could competitively disrupt a subset of the neurexin–neuroligin junctions consequently impairing the integrity of glutamatergic synapses. This might serve a molecular mechanism of excessive AChE induced neurodegeneration. PMID:24594013

  9. Interactions of AChE with Aβ Aggregates in Alzheimer’s Brain: Therapeutic Relevance of IDN 5706

    PubMed Central

    Carvajal, Francisco J.; Inestrosa, Nibaldo C.

    2011-01-01

    Acetylcholinesterase (AChE; EC 3.1.1.7) plays a crucial role in the rapid hydrolysis of the neurotransmitter acetylcholine, in the central and peripheral nervous system and might also participate in non-cholinergic mechanism related to neurodegenerative diseases. Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, amyloid-β (Aβ) peptide accumulation and synaptic alterations. We have previously shown that AChE is able to accelerate the Aβ peptide assembly into Alzheimer-type aggregates increasing its neurotoxicity. Furthermore, AChE activity is altered in brain and blood of Alzheimer’s patients. The enzyme associated to amyloid plaques changes its enzymatic and pharmacological properties, as well as, increases its resistant to low pH, inhibitors and excess of substrate. Here, we reviewed the effects of IDN 5706, a hyperforin derivative that has potential preventive effects on the development of AD. Our results show that treatment with IDN 5706 for 10 weeks increases brain AChE activity in 7-month-old double transgenic mice (APPSWE–PS1) and decreases the content of AChE associated with different types of amyloid plaques in this Alzheimer’s model. We concluded that early treatment with IDN 5706 decreases AChE–Aβ interaction and this effect might be of therapeutic interest in the treatment of AD. PMID:21949501

  10. Nature: A Substantial Source of Auspicious Substances with Acetylcholinesterase Inhibitory Action

    PubMed Central

    Orhan, Ilkay Erdogan

    2013-01-01

    Acetylcholinesterase (AChE) (EC 3.1.1.7) is an important enzyme that breaks down of acetylcholine in synaptic cleft in neuronal junctions. Inhibition of AChE is associated with treatment of several diseases such as Alzheimer’s disease (AD), myasthenia gravis, and glaucoma as well as the mechanisms of insecticide and anthelmintic drugs. Several AChE inhibitors are available in clinical use currently for the treatment of AD; however, none of them has ability, yet, to seize progress of the disease. Consequently, an extensive research has been going on finding new AChE inhibitors. In this sense, natural inhibitors have gained great attention due to their encouraging effects toward AChE. In this review, promising candidate molecules with marked AChE inhibition from both plant and animal sources will be underlined. PMID:24381529

  11. Two heparin-binding domains are present on the collagenic tail of asymmetric acetylcholinesterase.

    PubMed

    Deprez, P N; Inestrosa, N C

    1995-05-12

    The collagen-tailed form of acetylcholinesterase (AChE) binds to heparin and heparan sulfate proteoglycans. We have employed synthetic peptides corresponding to the central collagenic region of the tail of AChE, to identify the heparin-binding domains of the tail of asymmetric AChE. Two putative heparin-binding consensus sequences were localized in the collagenic tail. Peptides containing such sequences (P-(145-159) and P-(249-262)) were able to release asymmetric AChE bound to heparin-agarose. A triple mutation, Asn-Asp-Gly-Gly instead of Arg-His-Gly-Arg, completely abolishes the capacity of the peptide P-(145-159) to elute AChE from the heparin column. Our results suggest that the interaction between the collagen-tailed AChE and proteoglycans is mediated by clusters of basic residues that form two belts around the triple helix of the collagenic tail.

  12. Primary Investigation for the Mechanism of Biatractylolide from Atractylodis Macrocephalae Rhizoma as an Acetylcholinesterase Inhibitor

    PubMed Central

    Xie, Yong-Chao; Ning, Ning; Zhu, Li; Li, Dan-Ning

    2016-01-01

    Biatractylolide was isolated from ethyl acetate extract of dried Atractylodis Macrocephalae Rhizoma root by multistep chromatographic processing. Structure of biatractylolide was confirmed by 1H-NMR and 13C-NMR. The IC50 on acetylcholinesterase (AChE) activity was 6.5458 μg/mL when the control IC50 value of huperzine A was 0.0192 μg/mL. Molecular Docking Software (MOE) was used to discover molecular sites of action between biatractylolide and AChE protein by regular molecular docking approaches. Moreover, biatractylolide downregulated the expression of AChE of MEF and 293T cells in a dose-dependent manner. These results demonstrated that the molecular mechanisms of inhibitory activities of biatractylolide on AChE are not only through binding to AChE, but also via reducing AChE expression by inhibiting the activity of GSK3β. PMID:27642355

  13. Purification and characterization of acetylcholinesterase from desert cobra (Walterinnesia aegyptia) venom.

    PubMed

    Duhaiman, A S; Alhomida, A S; Rabbani, N; Kamal, M A; al-Jafari, A A

    1996-01-01

    Acetylcholinesterase (AChE) has been identified and purified from the venom of desert cobra (W aegyptia) to apparent homogeneity using a TSK G 3000 SW gel filtration column and a Mono Q anion-exchange column. AChE was purified to homogeneity as established by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The specific activity of AChE was 357 IU/mg with acetylthiocholine iodide as substrate. The denatured W aegyptia venom AChE displayed a molecular mass of 67000 +/- 3000 Da suggesting it was a single polypeptide. Isoelectric focusing of AChE revealed that the enzyme exists in different isoforms, with isoelectric points ranging between pH 7.4-7.9. The kinetic parameters (Km and Vmax) and IC50 of AChE inhibition by procaine, tetracaine and physostigmine were investigated in the present study.

  14. Acetylcholinesterase inhibitory effect of lignans isolated from Schizandra chinensis.

    PubMed

    Hung, Tran Manh; Na, MinKyun; Min, Byung Sun; Ngoc, Tran Minh; Lee, IkSoo; Zhang, XinFeng; Bae, KiHwan

    2007-06-01

    The hexane extract of the fruit of Schizandra chinensis (Schisandraceae) was found to show significant inhibition of the activity of acetylcholinesterase enzyme (AChE). In further studies, fourteen lignans were isolated, and evaluated for their inhibitory effect on AChE. The compounds having both aromatic methylenedioxy and hydroxyl groups on their cyclooctadiene ring, such as gomisin C (6), gomisin G (7), gomisin D (8), schisandrol B (11) and gomisin A (13), entirely inhibited AChE in dose dependent manners, with IC50 values of 6.71 +/- 0.53, 6.55 +/- 0.31, 7.84 +/- 0.62, 12.57 +/- 1.07 and 13.28 +/- 1.68 microM, respectively. These results indicate that the lignans could potentially be a potent class of AChE inhibitors.

  15. Acetylcholinesterase biosensor for carbaryl detection based on interdigitated array microelectrodes.

    PubMed

    Gong, Zhili; Guo, Yemin; Sun, Xia; Cao, Yaoyao; Wang, Xiangyou

    2014-10-01

    In this study, an acetylcholinesterase (AChE) biosensor with superior accuracy and sensitivity was successfully developed based on interdigitated array microelectrodes (IAMs). IAMs have a series of parallel microband electrodes with alternating microbands connected together. Chitosan was used as the enzyme immobilization material, and AChE was used as the model enzyme for carbaryl detection to fabricate AChE biosensor. Electrochemical impedance spectroscopy was used in conjunction with the fabricated biosensor to detect pesticide residues. Based on the inhibition of pesticides on the AChE activity, using carbaryl as model compounds, the biosensor exhibited a wide range, low detection limit, and high stability. Moreover, the biosensor can also be used as a new promising tool for pesticide residue analysis.

  16. Centrally acting oximes in reactivation of tabun-phosphoramidated AChE.

    PubMed

    Kovarik, Zrinka; Maček, Nikolina; Sit, Rakesh K; Radić, Zoran; Fokin, Valery V; Barry Sharpless, K; Taylor, Palmer

    2013-03-25

    Organophosphates (OP) inhibit acetylcholinesterase (AChE, EC 3.1.1.7), both in peripheral tissues and central nervous system (CNS), causing adverse and sometimes fatal effects due to the accumulation of neurotransmitter acetylcholine (ACh). The currently used therapy, focusing on the reactivation of inhibited AChE, is limited to peripheral tissues because commonly used quaternary pyridinium oxime reactivators do not cross the blood brain barrier (BBB) at therapeutically relevant levels. A directed library of thirty uncharged oximes that contain tertiary amine or imidazole protonable functional groups that should cross the BBB as unionized species was tested as tabun-hAChE conjugate reactivators along with three reference oximes: DAM (diacetylmonoxime), MINA (monoisonitrosoacetone), and 2-PAM. The oxime RS150D [N-((1-(3-(2-((hydroxyimino)methyl)-1H-imidazol-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide] was highlighted as the most promising reactivator of the tabun-hAChE conjugate. We also observed that oximes RS194B [N-(2-(azepan-1-yl)ethyl)-2-(hydroxyimino)acetamide] and RS41A [2-(hydroxyimino)-N-(2-(pyrrolidin-1-yl)ethyl)acetamide], which emerged as lead uncharged reactivators of phosphylated hAChE with other OPs (sarin, cyclosarin and VX), exhibited only moderate reactivation potency for tabun inhibited hAChE. This implies that geometry of oxime access to the phosphorus atom conjugated to the active serine is an important criterion for efficient reactivation, along with the chemical nature of the conjugated moiety: phosphorate, phosphonate, or phosphoramidate. Moreover, modification of the active center through mutagenesis enhances the rates of reactivation. The phosphoramidated-hAChE choline-binding site mutant Y337A showed three-times enhanced reactivation capacity with non-triazole imidazole containing aldoximes (RS113B, RS113A and RS115A) and acetamide derivative (RS194B) than with 2PAM.

  17. Design, synthesis, and characterization of novel, nonquaternary reactivators of GF-inhibited human acetylcholinesterase.

    PubMed

    McHardy, Stanton F; Bohmann, Jonathan A; Corbett, Michael R; Campos, Bismarck; Tidwell, Michael W; Thompson, Paul Marty; Bemben, Chris J; Menchaca, Tony A; Reeves, Tony E; Cantrell, William R; Bauta, William E; Lopez, Ambrosio; Maxwell, Donald M; Brecht, Karen M; Sweeney, Richard E; McDonough, John

    2014-04-01

    The goal of this research was to identify structurally novel, non-quaternarypyridinium reactivators of GF (cyclosarin)-inhibited hAChE that possess the capacity to mediate in vitro reactivation of GF-inhibited human acetylcholinesterase (hAChE). New compounds were designed, synthesized and assessed in GF-inhibited hAChE assays. Structure activity relationships for AChE binding and reactivation of GF-inhibited hAChE were developed. Lead compounds from two different chemical series, represented by compounds 17 and 38, displayed proficient in vitro reactivation of GF-inhibited hAChE, while also possessing low inhibition of native enzyme.

  18. Acetylcholinesterase inhibitors and Gulf War illnesses

    PubMed Central

    Golomb, Beatrice Alexandra

    2008-01-01

    Increasing evidence suggests excess illness in Persian Gulf War veterans (GWV) can be explained in part by exposure of GWV to organophosphate and carbamate acetylcholinesterase inhibitors (AChEis), including pyridostigmine bromide (PB), pesticides, and nerve agents. Evidence germane to the relation of AChEis to illness in GWV was assessed. Many epidemiological studies reported a link between AChEi exposure and chronic symptoms in GWV. The link is buttressed by a dose–response relation of PB pill number to chronic symptoms in GWV and by a relation between avidity of AChEi clearance and illness, based on genotypes, concentrations, and activity levels of enzymes that detoxify AChEis. Triangulating evidence derives from studies linking occupational exposure to AChEis to chronic health symptoms that mirror those of ill GWV. Illness is again linked to lower activity of AChEi detoxifying enzymes and genotypes conferring less-avid AChEi detoxification. AChEi exposure satisfies Hill's presumptive criteria for causality, suggesting this exposure may be causally linked to excess health problems in GWV. PMID:18332428

  19. Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma.

    PubMed

    Castillo-González, Ana Cristina; Pelegrín-Hernández, Juan Pablo; Nieto-Cerón, Susana; Madrona, Antonio Piñero; Noguera, José Antonio; López-Moreno, María Fuensanta; Rodríguez-López, José Neptuno; Vidal, Cecilio J; Hellín-Meseguer, Diego; Cabezas-Herrera, Juan

    2015-11-01

    Previous reports have demonstrated that a non-neuronal cholinergic system is expressed aberrantly in airways. A proliferative effect is exerted directly by cholinergic agonists through the activation of nicotinic and muscarinic receptors. In cancer, particularly those related with smoking, the mechanism through which tumour cells respond to aberrantly activated cholinergic signalling is a key question. Fifty paired pieces of larynx squamous cell carcinoma and adjacent non-cancerous tissue were compared in terms of their acetylcholinesterase activity (AChE). The AChE activity in non-cancerous tissues (0.248 ± 0.030 milliunits per milligram of wet tissue; mU/mg) demonstrates that upper respiratory tissues express sufficient AChE activity for controlling the level of acetylcholine (ACh). In larynx carcinomas, the AChE activity decreased to 0.157 ± 0.024 mU/mg (p=0.009). Larynx cancer patients exhibiting low ACh-degrading enzymatic activity had a significantly shorter overall survival (p=0.031). Differences in the mRNA levels of alternatively spliced AChE isoforms and molecular compositions were noted between glottic and supraglottic cancers. Our results suggest that the low AChE activity observed in larynx squamous cell carcinoma may be useful for predicting the outcome of patients.

  20. Flavonoids induce the expression of acetylcholinesterase in cultured osteoblasts.

    PubMed

    Xu, Miranda L; Bi, Cathy W C; Kong, Ava Y Y; Dong, Tina T X; Wong, Yung H; Tsim, Karl W K

    2016-11-25

    Flavonoids, a group of natural compounds mainly derived from plants, are known to possess osteogenic effects in bone cells. Here, we aimed to test if flavonoid could induce a cholinergic enzyme, acetylcholinesterase (AChE), as well as bone differentiation. In cultured rat osteoblasts, twenty flavonoids, deriving from Chinese herbs and having known induction of alkaline phosphatase (ALP(1)) expression, were tested for its induction activity on AChE expression. Eleven flavonoids showed the induction, and five of them had robust activation of AChE expression, including baicalin, calycosin, genistin, hyperin and pratensein: the induction of AChE included the levels of mRNA, protein and enzymatic activity. Moreover, the flavonoid-induced AChE expression in cultured osteoblast was in proline-rich membrane anchor (PRiMA)-linked tetrameric globular form (G4) only. In parallel, the expression of PRiMA was also induced by the application of flavonoids. The flavonoid-induced AChE in the cultures was not affected by estrogen receptor blocker, ICI 182,780. Taken together, the induction of PRiMA-linked AChE in osteoblast should be independent to classical estrogen signaling pathway.

  1. Identification of two acetylcholinesterases in Pardosa pseudoannulata and the sensitivity to insecticides.

    PubMed

    Zhang, Yixi; Shao, Ying; Jiang, Feng; Li, Jian; Liu, Zewen

    2014-03-01

    Pardosa pseudoannulata is an important predatory enemy against insect pests, such as rice planthoppers and leafhoppers. In order to understand the insecticide selectivity between P. pseudoannulata and insect pests, two acetylcholinesterase genes, Pp-ace1 and Pp-ace2, were cloned from this natural enemy. The putative proteins encoded by Pp-ace1 and Pp-ace2 showed high similarities to insect AChE1 (63% to Liposcelis entomophila AChE1) and AChE2 (36% to Culex quinquefasciatus AChE2) with specific functional motifs, which indicated that two genes might encode AChE1 and AChE2 proteins respectively. The recombinant proteins by expressing Pp-ace1 and Pp-ace2 genes in insect sf9 cells showed high AChE activities. The kinetic parameters, Vmax and Km, of two recombinant AChE proteins were significantly different. The sensitivities to six insecticides were determined in two recombinant AChEs. Pp-AChE1 was more sensitive to all tested insecticides than Pp-AChE2, such as fenobucarb (54 times in Ki ratios), isoprocarb (31 times), carbaryl (13 times) and omethoate (6 times). These results indicated that Pp-AChE1 might be the major synaptic enzyme in the spider. By sequence comparison of P. pseudoannulata and insect AChEs, the key amino acid differences at or close to the functional sites were found. The locations of some key amino acid differences were consistent with the point mutation sites in insect AChEs that were associated with insecticide resistance, such as Phe331 in Pp-AChE2 corresponding to Ser331Phe mutation in Myzus persicae and Aphis gossypii AChE2, which might play important roles in insecticide selectivity between P. pseudoannulata and insect pests. Of course, the direct evidences are needed through further studies.

  2. Molecular Cloning and Characterization of an Acetylcholinesterase cDNA in the Brown Planthopper, Nilaparvata lugens

    PubMed Central

    Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

    2010-01-01

    A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain. PMID:20874389

  3. Study of Inhibition, Reactivation and Aging Processes of Pesticides Using Graphene Nanosheets/Gold Nanoparticles-Based Acetylcholinesterase Biosensor

    SciTech Connect

    Zhang, Lin; Long, Linjuan; Zhang, Weiying; Du, Dan; Lin, Yuehe

    2012-09-10

    Organophosphate (OP) and carbamate pesticides exert their toxicity via attacking the hydroxyl moiety of serine in the 'active site' of acetylcholinesterase (AChE). In this paper we developed a stable AChE biosensor based on self-assembling AChE to graphene nanosheet (GN)-gold nanoparticles (AuNPs) nanocomposite electrode for investigation of inhibition, reactivation and aging processes of different pesticides. It is confirmed that pesticides can inhibit AChE in a short time. OPs poisoning is treatable with oximes while carbarmates exposure is insensitive to oximes. The proposed electrochemical approach thus provides a new simple tool for comparison of pesticide sensitivity and guide of therapeutic intervention.

  4. Development of a 3D-QSAR model for acetylcholinesterase inhibitors using a combination of fingerprint, docking, and structure-based pharmacophore approaches - Conference Abstract

    EPA Science Inventory

    Acetylcholinesterase (AChE), a serine hydrolase vital for regulating the neurotransmitter acetylcholine in animals, has been used as a target for drugs and pesticides. With the increasing availability of AChE crystal structures, with or without ligands bound, structure-based appr...

  5. Development of 3D-QSAR model for acetylcholinesterase inhibitors using a combination of fingerprint, molecular docking, and structure-based pharmacophore approaches

    EPA Science Inventory

    Acetylcholinesterase (AChE), a serine hydrolase vital for regulating the neurotransmitter acetylcholine in animals, has been used as a target for drugs and pesticides. With the increasing availability of AChE crystal structures, with or without ligands bound, structure-based appr...

  6. Subchronic effects following a single sarin exposure on blood-brain and blood-testes barrier permeability, acetylcholinesterase, and acetylcholine receptors in the central nervous system of rat: a dose-response study.

    PubMed

    Jones, K H; Dechkovskaia, A M; Herrick, E A; Abdel-Rahman, A A; Khan, W A; Abou-Donia, M B

    2000-12-29

    Subchronic neurotoxic effects of sarin (O-isopropyl methylphosphonofluoridate) treatment at various doses in male Sprague Dawley rats were studied. The animals were treated with a single intramuscular (im) injection of 0.01, 0.1, 0.5, or 1 x LD50 (100 microg/kg). The animals were maintained for 90 d thereafter. [3H]Hexamethonium iodide was used to monitor the changes in blood-brain barrier (BBB) permeability in cortex, brainstem, midbrain, and cerebellum. Brainstem exhibited a significant decrease (approximately 58% of control) in uptake of [3H]hexamethonium iodide at 1 x LD50 dose. No significant changes were observed in BBB permeability in cortex, midbrain, and cerebellum at any dose. Plasma butyrylcholinesterase (BChE) activity remained unchanged, reflecting recovery of the enzyme activity from the initial inhibition following single exposure of 1 x LD50 sarin. Acetylcholinesterase (AChE) activity in the cortex remained inhibited (approximately 29%), whereas in the brainstem there was an increase (approximately 20%) at 1 x LD50 dose of sarin. The m2-selective muscarinic acetylcholine receptor (m2-mAChR) ligand binding was inhibited significantly at 1 x LD50 in the cortex, whereas brainstem showed significantly increased (approximately 45%) ligand binding at 1 x LD50 dose. Nicotinic acetylcholine receptor (nAChR), on the other hand, showed a biphasic response in ligand binding in the cortex with a decrease (approximately 30%) at 0.01 x LD50 but an increase (approximately 40%) at 1 x LD5O. Brainstem did not show any significant change in nAChR ligand binding. These results suggest that single exposure of sarin could lead to changes that may play an important role in neuropathological abnormalities in the central nervous system.

  7. Computer Image Analysis of Histochemically-Labeled Acetylcholinesterase.

    DTIC Science & Technology

    1984-11-30

    image analysis on conjunction with histochemical techniques to describe the distribution of acetylcholinesterase (AChE) activity in nervous and muscular tissue in rats treated with organophosphates (OPs). The objective of the first year of work on this remaining 2 years. We began by adopting a version of the AChE staining method as modified by Hanker, which consistent with the optical properties of our video system. We wrote computer programs for provide a numeric quantity which represents the degree of staining in a tissue section. The staining was calibrated by

  8. Effect of isoquinoline alkaloids from two Hippeastrum species on in vitro acetylcholinesterase activity.

    PubMed

    Pagliosa, L B; Monteiro, S C; Silva, K B; de Andrade, J P; Dutilh, J; Bastida, J; Cammarota, M; Zuanazzi, J A S

    2010-07-01

    The treatment of neurological disorders and neurodegenerative diseases is related to the levels of acetylcholine (ACh) through the inhibition of acetylcholinesterase (AChE). Galanthamine, an important alkaloid isolated from the Amaryllidaceae family, is approved for the pharmacological treatment of Alzheimer's disease (AD) and acts by inhibiting the acetylcholinesterase (AChE) activity. In the present study, Ellman's method was used to verify the inhibition of AChE activity of some isoquinolines alkaloids such as galanthamine, montanine, hippeastrine and pretazettine. At the concentrations 1mM, 500 microm and 100 microm, galanthamine presented an AChE inhibition higher than 90%. Montanine inhibited, in a dose-dependent manner, more than 50% of the enzyme at 1mM concentration. With the concentrations 500 microm and 100 microm, 30-45% of AChE activity inhibition was detected. The alkaloids hippeastrine and pretazettine presented no significant inhibition of the AChE activity. The results demonstrate that montanine significantly inhibits AChE activity at the tested concentrations, suggesting the necessity of further investigations on this alkaloid use in treating neurological disorders.

  9. Protection of rhesus monkeys against Soman and prevention of performance decrement by pretreatment with acetylcholinesterase. (Reannouncement with new availability information)

    SciTech Connect

    Maxwell, D.M.; Castro, C.A.; De La Hoz, D.M.; Gentry, M.K.; Gold, M.B.

    1992-12-31

    The ability of acetylcholinesterase from fetal bovine serum (FBS AChE) to protect against soman, a highly toxic organophosphorus (OP) compound, was tested in rhesus monkeys. Intravenous administration of FBS AChE produced a minimal behavioral effect on the serial probe recognition task, a sensitive test of cognitive function and short-term memory. Pharmacokinetic studies of injected FBS AChE indicated a plasma half-life of 40 hr for FBS AChE in monkeys. Both in vitro and in vivo titration of FBS AChE with soman produced a 1:1 stoichiometry between organophosphate-inhibited FBS AChE and the cumulative dose of the toxic stereoisomers of soman. Administration of FBS AChE protected monkeys against the lethal effects of up to 2.7 LD50 of soman and prevented any signs of organophosphate intoxication, e.g., excessive secretions, respiratory depression, muscle fasciculations, or convulsions. In addition, monkeys pretreated with FBS AChE were devoid of any behavioral incapacitation after soman challenge, as measured by the serial probe recognition task. Compared to the current multicomponent drug treatment against soman, which does not prevent the signs or the behavioral deficits resulting from OP intoxication, use of FBS AChE as a single pretreatment drug provides significantly effective protection against both the lethal and the behavioral effects of soman.... Pretreatment, Nonhuman primate, Performance decrements, Acetylcholinesterase, Soman, Nerve agents.

  10. Comparison of oxime reactivation and aging of nerve agent-inhibited monkey and human acetylcholinesterases.

    PubMed

    Luo, Chunyuan; Tong, Min; Maxwell, Donald M; Saxena, Ashima

    2008-09-25

    Non-human primates are valuable animal models that are used for the evaluation of nerve agent toxicity as well as antidotes and results from animal experiments are extrapolated to humans. It has been demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited acetylcholinesterase (AChE). If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the aging and reactivation of human and different monkey (Rhesus, Cynomolgus, and African Green) AChEs inhibited by GF, GD, and VR. The oximes examined include the traditional oxime 2-PAM, two H-oximes HI-6 and HLo-7, and the new candidate oxime MMB4. Results indicate that oxime reactivation of all three monkey AChEs was very similar to human AChE. The maximum difference in the second-order reactivation rate constant between human and three monkey AChEs or between AChEs from different monkey species was 5-fold. Aging rate constants of GF-, GD-, and VR-inhibited monkey AChEs were very similar to human AChE except for GF-inhibited monkey AChEs, which aged 2-3 times faster than the human enzyme. The results of this study suggest that all three monkey species are suitable animal models for nerve agent antidote evaluation since monkey AChEs possess similar biochemical/pharmacological properties to human AChE.

  11. Nanomaterials-Based Optical Techniques for the Detection of Acetylcholinesterase and Pesticides

    PubMed Central

    Xia, Ning; Wang, Qinglong; Liu, Lin

    2015-01-01

    The large amount of pesticide residues in the environment is a threat to global health by inhibition of acetylcholinesterase (AChE). Biosensors for inhibition of AChE have been thus developed for the detection of pesticides. In line with the rapid development of nanotechnology, nanomaterials have attracted great attention and have been intensively studied in biological analysis due to their unique chemical, physical and size properties. The aim of this review is to provide insight into nanomaterial-based optical techniques for the determination of AChE and pesticides, including colorimetric and fluorescent assays and surface plasmon resonance. PMID:25558991

  12. Toxicological and Biochemical Characterizations of AChE in Phosalone-Susceptible and Resistant Populations of the Common Pistachio Psyllid, Agonoscena pistaciae

    PubMed Central

    Alizadeh, Ali; Talebi-Jahromi, Khalil; Hosseininaveh, Vahid; Ghadamyari, Mohammad

    2014-01-01

    The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (KM) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively

  13. Brain acetylcholinesterase and its molecular forms in a precocial murid, Acomys cahirinus, and rat during post-natal development.

    PubMed

    Michalek, H; Pintor, A; Fortuna, S; Bisso, G M

    1984-01-01

    Brain acetylcholinesterase (AChE) and its molecular forms of a precocial murid, Acomys cahirinus, characterized by a large hippocampus, were measured during post-natal development and compared with rat. The activity of soluble AChE in Acomys increased slightly up to 4 weeks after birth. The total AChE activity increased somewhat more but, in rats, this increase was still greater. Three main molecular forms of AChE were separated by 7.5% polyacrylamide gel electrophoresis. Their close similarity to the rat AChE forms was assessed by gradient polyacrylamide gel electrophoresis and electrofocusing. Maturation of these forms, i.e., conversion of simple into more complex forms in the soluble fraction of AChE was, however, considerably delayed reaching only after 4 weeks the pattern comparable to that of rat.

  14. [A comparison of the efficacy of the reactivators of acetylcholinesterase inhibited with tabun].

    PubMed

    Cabal, J; Kuca, K; Jun, D; Bajgar, J; Hrabinová, M

    2005-07-01

    The nerve agent tabun inhibits acetylcholinesterase (AChE; EC 3.1.1.7) by the formation of a covalent bond with the enzyme. Afterwards, AChE is not able to fulfil its role in the organism and subsequently cholinergic crisis occurs. AChE reactivators (pralidoxime, obidoxime and HI-6) as causal antidotes are used for the cleavage of the bond between the enzyme and nerve agent. Unfortunately, their potency for reactivation of tabun-inhibited AChE is poor. The aim of the study was to choose the most potent reactivator of tabun-inhibited AChE. We have tested eight AChE reactivators--pralidoxime, obidoxime, trimedoxime, HI-6, methoxime, Hlö-7 and our newly synthesized oximes K027 and K048. All reactivators were tested using our standard in vitro reactivation test (pH 8, 25 degrees C, time of inhibition by the nerve agent 30 minutes, time of reactivation by AChE reactivator 10 minutes). According to our results, only trimedoxime was able to achieve 50% reactivation potency. However, this relatively high potency was achieved at high oxime concentration (10(-2) M). At a lower concentration of 10(-4) M (the probably attainable concentration in vivo), four AChE reactivators (trimedoxime, obidoxime, K027, and K048) were able to reactivate AChE inhibited by tabun reaching from 10 to 18%.

  15. Evolutionary origin and status of two insect acetylcholinesterases and their structural conservation and differentiation.

    PubMed

    Cha, Deok Jea; Lee, Si Hyeock

    2015-01-01

    Acetylcholinesterase (AChE) plays a pivotal role in synaptic transmission in the cholinergic nervous system of most animals, including insects. Insects possess duplicated AChE gene loci (ace1 vs. ace2) encoding two distinct AChEs (AChE1 and AChE2). A phylogenetic analysis suggested that the last common ancestor of two aces shared its origin with Platyhelminthes. In addition, the ace duplication event likely occurred after the divergence of Protostomian but before the split of Ecdysozoa. The ace1 lineage exhibited a significantly lower evolutionary rate (d and dN/dS ratio) than the ace2 lineage, suggesting that the ace1 lineage has retained the essential function of synaptic transmission following its duplication. Therefore, the putative functional transition from ace1 to ace2 observed in some Hymenopteran insects appears to be a local and relatively recent event. The amino acid sequence comparison and three-dimensional modeling of insect AChEs identified a few consistent differences in the amino acid residues in functionally crucial domains between two AChEs, which are likely responsible for the functional differentiation between two AChEs. A unique amino acid substitution causing a dramatic reduction in the catalytic activity of AChE1 in some Hymenopteran insects was suggested to be responsible for the aforementioned functional transition of ace.

  16. Effect of local acetylcholinesterase inhibition on sweat rate in humans

    NASA Technical Reports Server (NTRS)

    Shibasaki, M.; Crandall, C. G.

    2001-01-01

    ACh is the neurotransmitter responsible for increasing sweat rate (SR) in humans. Because ACh is rapidly hydrolyzed by acetylcholinesterase (AChE), it is possible that AChE contributes to the modulation of SR. Thus the primary purpose of this project was to identify whether AChE around human sweat glands is capable of modulating SR during local application of various concentrations of ACh in vivo, as well as during a heat stress. In seven subjects, two microdialysis probes were placed in the intradermal space of the forearm. One probe was perfused with the AChE inhibitor neostigmine (10 microM); the adjacent membrane was perfused with the vehicle (Ringer solution). SR over both membranes was monitored via capacitance hygrometry during microdialysis administration of various concentrations of ACh (1 x 10(-7)-2 M) and during whole body heating. SR was significantly greater at the neostigmine-treated site than at the control site during administration of lower concentrations of ACh (1 x 10(-7)-1 x 10(-3) M, P < 0.05), but not during administration of higher concentrations of ACh (1 x 10(-2)-2 M, P > 0.05). Moreover, the core temperature threshold for the onset of sweating at the neostigmine-treated site was significantly reduced relative to that at the control site. However, no differences in SR were observed between sites after 35 min of whole body heating. These results suggest that AChE is capable of modulating SR when ACh concentrations are low to moderate (i.e., when sudomotor activity is low) but is less effective in governing SR after SR has increased substantially.

  17. Induction of plasma acetylcholinesterase activity in mice challenged with organophosphorus poisons

    SciTech Connect

    Duysen, Ellen G.; Lockridge, Oksana

    2011-09-01

    The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher than the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase. - Highlights: > Acetylcholinesterase activity is induced by organophosphorus agents. > AChE induction is related to apoptosis. > Induction of AChE activity by OP is independent of BChE.

  18. Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To construct the Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE), the gene for the anchor protein (AGa1) was obtained from Saccharomyces cerevisiae and was fused with the modified Bombyx mori acetylcholinesterase gene (bmace) and transformed int...

  19. Effect of metoclopramide and ranitidine on the inhibition of human AChE by VX in vitro.

    PubMed

    Bartling, A; Thiermann, H; Szinicz, L; Worek, F

    2005-01-01

    The repeated misuse of highly toxic organophosphorus-type (OP) chemical warfare agents ('nerve agents') emphasizes the necessity for the development of effective medical countermeasures. The standard treatment with atropine and acetylcholinesterase (AChE) reactivators ('oximes') is considered to be ineffective with certain nerve agents due to low oxime efficacy. Therefore, pretreatment with carbamate-type compounds, e.g. pyridostigmine, was recommended to improve antidotal efficacy. Recently, the clinically used reversible AChE inhibitors metoclopramide (MCP) and ranitidine (RAN) were shown to exhibit some protective effect against the OP pesticide paraoxon in vitro and in vivo. The present study was undertaken to investigate a potential protective effect of MCP and RAN against inhibition of human AChE by the nerve agent VX (O-ethyl S-[2-(diisopropylamino)ethyl)methylphosphonothioate). Hemoglobin-free human erythrocyte membranes were incubated with various, human relevant MCP (0.5-2 microm) and RAN (0.5-5 microm) concentrations starting 1 min before addition of VX (1-40 nm). Both compounds failed to increase VX IC(50) values. In addition, human AChE was incubated with higher than human relevant therapeutic concentrations of MCP (1 microm-1 mm) and RAN (1 microm-2.0 mm) and inhibited by 40 nm VX. At concentrations higher than 100 microm MCP and RAN caused a concentration dependent increase of residual AChE activity 15 min after addition of VX. These data indicate that MCP and RAN may be ineffective in protecting human AChE against inhibition by the nerve agent VX at human relevant doses.

  20. Design, synthesis and structure-activity relationships of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease.

    PubMed

    Toda, Narihiro; Tago, Keiko; Marumoto, Shinji; Takami, Kazuko; Ori, Mayuko; Yamada, Naho; Koyama, Kazuo; Naruto, Shunji; Abe, Kazumi; Yamazaki, Reina; Hara, Takao; Aoyagi, Atsushi; Abe, Yasuyuki; Kaneko, Tsugio; Kogen, Hiroshi

    2003-05-01

    We have designed and synthesized a dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT) as a novel class of treatment drugs for Alzheimer's disease on the basis of a hypothetical model of the AChE active site. Dual inhibitions of AChE and SERT would bring about greater therapeutic effects than AChE inhibition alone and avoid adverse peripheral effects caused by excessive AChE inhibition. Compound (S)-6j exhibited potent inhibitory activities against AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM). Furthermore, (S)-6j showed inhibitory activities of both AChE and SERT in mice brain following oral administration.

  1. A conformational restriction approach to the development of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease.

    PubMed

    Toda, Narihiro; Tago, Keiko; Marumoto, Shinji; Takami, Kazuko; Ori, Mayuko; Yamada, Naho; Koyama, Kazuo; Naruto, Shunji; Abe, Kazumi; Yamazaki, Reina; Hara, Takao; Aoyagi, Atsushi; Abe, Yasuyuki; Kaneko, Tsugio; Kogen, Hiroshi

    2003-10-01

    Alzheimer's disease (AD) has been treated with acetylcholinesterase (AChE) inhibitors such as donepezil. However, the clinical usefulness of AChE inhibitors is limited mainly due to their adverse peripheral effects. Depression seen in AD patients has been treated with serotonin transporter (SERT) inhibitors. We considered that combining SERT and AChE inhibition could improve the clinical usefulness of AChE inhibitors. In a previous paper, we found a potential dual inhibitor, 1, of AChE (IC50=101 nM) and SERT (IC50=42 nM), but its AChE inhibition activity was less than donepezil (IC50=10 nM). Here, we report the conformationally restricted (R)-18a considerably enhanced inhibitory activity against AChE (IC50=14 nM) and SERT (IC50=6 nM).

  2. Identification and Biochemical Properties of Two New Acetylcholinesterases in the Pond Wolf Spider (Pardosa pseudoannulata)

    PubMed Central

    Meng, Xiangkun; Li, Chunrui; Xiu, Chunli; Zhang, Jianhua; Li, Jingjing; Huang, Lixin; Zhang, Yixi; Liu, Zewen

    2016-01-01

    Acetylcholinesterase (AChE), an important neurotransmitter hydrolase in both invertebrates and vertebrates, is targeted by organophosphorus and carbamate insecticides. In this study, two new AChEs were identified in the pond wolf spider Pardosa pseudoannulata, an important predatory natural enemy of several insect pests. In total, four AChEs were found in P. pseudoannulata (including two AChEs previously identified in our laboratory). The new putative AChEs PpAChE3 and PpAChE4 contain most of the common features of the AChE family, including cysteine residues, choline binding sites, the conserved sequence ‘FGESAG’ and conserved aromatic residues but with a catalytic triad of ‘SDH’ rather than ‘SEH’. Recombinant enzymes expressed in Sf9 cells showed significant differences in biochemical properties compared to other AChEs, such as the optimal pH, substrate specificity, and catalytic efficiency. Among three test substrates, PpAChE1, PpAChE3 and PpAChE4 showed the highest catalytic efficiency (Vmax/KM) for ATC (acetylthiocholine iodide), with PpAChE3 exhibiting a clear preference for ATC based on the VmaxATC/VmaxBTC ratio. In addition, the four PpAChEs were more sensitive to the AChE-specific inhibitor BW284C51, which acts against ATC hydrolysis, than to the BChE-specific inhibitor ISO-OMPA, which acts against BTC hydrolysis, with at least a 8.5-fold difference in IC50 values for each PpAChE. PpAChE3, PpAChE4, and PpAChE1 were more sensitive than PpAChE2 to the tested Carb insecticides, and PpAChE3 was more sensitive than the other three AChEs to the tested OP insecticides. Based on all the results, two new functional AChEs were identified from P. pseudoannulata. The differences in AChE sequence between this spider and insects enrich our knowledge of invertebrate AChE diversity, and our findings will be helpful for understanding the selectivity of insecticides between insects and natural enemy spiders. PMID:27337188

  3. Identification and Biochemical Properties of Two New Acetylcholinesterases in the Pond Wolf Spider (Pardosa pseudoannulata).

    PubMed

    Meng, Xiangkun; Li, Chunrui; Xiu, Chunli; Zhang, Jianhua; Li, Jingjing; Huang, Lixin; Zhang, Yixi; Liu, Zewen

    2016-01-01

    Acetylcholinesterase (AChE), an important neurotransmitter hydrolase in both invertebrates and vertebrates, is targeted by organophosphorus and carbamate insecticides. In this study, two new AChEs were identified in the pond wolf spider Pardosa pseudoannulata, an important predatory natural enemy of several insect pests. In total, four AChEs were found in P. pseudoannulata (including two AChEs previously identified in our laboratory). The new putative AChEs PpAChE3 and PpAChE4 contain most of the common features of the AChE family, including cysteine residues, choline binding sites, the conserved sequence 'FGESAG' and conserved aromatic residues but with a catalytic triad of 'SDH' rather than 'SEH'. Recombinant enzymes expressed in Sf9 cells showed significant differences in biochemical properties compared to other AChEs, such as the optimal pH, substrate specificity, and catalytic efficiency. Among three test substrates, PpAChE1, PpAChE3 and PpAChE4 showed the highest catalytic efficiency (Vmax/KM) for ATC (acetylthiocholine iodide), with PpAChE3 exhibiting a clear preference for ATC based on the VmaxATC/VmaxBTC ratio. In addition, the four PpAChEs were more sensitive to the AChE-specific inhibitor BW284C51, which acts against ATC hydrolysis, than to the BChE-specific inhibitor ISO-OMPA, which acts against BTC hydrolysis, with at least a 8.5-fold difference in IC50 values for each PpAChE. PpAChE3, PpAChE4, and PpAChE1 were more sensitive than PpAChE2 to the tested Carb insecticides, and PpAChE3 was more sensitive than the other three AChEs to the tested OP insecticides. Based on all the results, two new functional AChEs were identified from P. pseudoannulata. The differences in AChE sequence between this spider and insects enrich our knowledge of invertebrate AChE diversity, and our findings will be helpful for understanding the selectivity of insecticides between insects and natural enemy spiders.

  4. Buprofezin inhibits acetylcholinesterase activity in B-biotype Bemisia tabaci.

    PubMed

    Cottage, Emma L A; Gunning, Robin V

    2006-01-01

    B-biotype Bemisia tabaci is a severe insect pest worldwide in many ornamental, agricultural, and horticultural industries. Control of this insect is hampered by resistance to many acetylcholinesterase (AChE)-inhibiting insecticides, such as organophosphates and carbamates. Consequently, insect growth regulators such as buprofezin, which act by inhibiting chitin synthesis, are being investigated for use against B-biotype B. tabaci in Australia. This study discusses the effects of buprofezin on B. tabaciAChE.

  5. Inhibition of AChE by malathion and some structurally similar compounds.

    PubMed

    Krstić, Danijela Z; Colović, Mirjana; Kralj, Mojca Bavcon; Franko, Mladen; Krinulović, Katarina; Trebse, Polonca; Vasić, Vesna

    2008-08-01

    Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. KI, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k3, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. KI values of 1.3 x 10(-4) M(-1), 5.6 x 10(-6) M(-1) and 7.2 x 10(-6)M(-1) were obtained for malathion, malaoxon and isomalathion, respectively. The IC50 values for free/immobilized AChE, (3.7 +/- 0.2) x 10(-4) M/(1.6 +/-0.1) x 10(-4), (2.4 +/- 0.3) x 10(-6)/(3.4 +/- 0.1) x 10(-6)M and (3.2 +/- 0.3) x 10(-6) M/(2.7 +/- 0.2) x 10(-6) M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 x 10(-7) M/2 x 10(-7) M, 2 x 10(-7) M/3 x 10(-7)M and 2 x 10(-7) M/4.5 x 109-7) M), while an

  6. Acetylcholinesterases of blood-feeding flies and ticks.

    PubMed

    Temeyer, Kevin B; Tuckow, Alexander P; Brake, Danett K; Li, Andrew Y; Pérez de León, Adalberto A

    2013-03-25

    Acetylcholinesterase (AChE) is the biochemical target of organophosphate (OP) and carbamate pesticides for invertebrates, vertebrate nerve agents, and AChE inhibitors used to reduce effects of Alzheimer's disease. Organophosphate pesticides (OPs) are widely used to control blood-feeding arthropods, including biting flies and ticks. However, resistance to OPs in pests affecting animal and human health has compromised control efficacy. OP resistance often results from mutations producing an OP-insensitive AChE. Our studies have demonstrated production of OP-insensitive AChEs in biting flies and ticks. Complementary DNA (cDNA) sequences encoding AChEs were obtained for the horn fly, stable fly, sand fly, and the southern cattle tick. The availability of cDNA sequences enables the identification of mutations, expression and characterization of recombinant proteins, gene silencing for functional studies, as well as in vitro screening of novel inhibitors. The southern cattle tick expresses at least three different genes encoding AChE in their synganglion, i.e. brain. Gene amplification for each of the three known cattle tick AChE genes and expression of multiple alleles for each gene may reduce fitness cost associated with OP-resistance. AChE hydrolyzes the neurotransmitter, acetylcholine, but may have additional roles in physiology and development. The three cattle tick AChEs possess significantly different biochemical properties, and are expressed in neural and non-neural tissues, which suggest separation of structure and function. The remarkable complexity of AChEs in ticks suggested by combining genomic data from Ixodes scapularis with our genetic and biochemical data from Rhipicephalus microplus is suggestive of previously unknown gene duplication and diversification. Comparative studies between invertebrate and vertebrate AChEs could enhance our understanding of structure-activity relationships. Research with ticks as a model system offers the opportunity to

  7. Evaluation of the Toxicity, AChE Activity and DNA Damage Caused by Imidacloprid on Earthworms, Eisenia fetida.

    PubMed

    Wang, Kai; Qi, Suzhen; Mu, Xiyan; Chai, Tingting; Yang, Yang; Wang, Dandan; Li, Dongzhi; Che, Wunan; Wang, Chengju

    2015-10-01

    Imidacloprid is a well-known pesticide and it is timely to evaluate its toxicity to earthworms (Eisenia fetida). In the present study, the effect of imidacloprid on reproduction, growth, acetylcholinesterase (AChE) and DNA damage in earthworms was assessed using an artificial soil medium. The median lethal concentration (LC50) and the median number of hatched cocoons (EC50) of imidacloprid to earthworms was 3.05 and 0.92 mg/kg respectively, the lowest observed effect concentration of imidacloprid about hatchability, growth, AChE activity and DNA damage was 0.02, 0.5, 0.1 and 0.5 mg/kg, respectively.

  8. Can Salivary Acetylcholinesterase be a Diagnostic Biomarker for Alzheimer?

    PubMed Central

    Bakhtiari, Sedigheh; Moghadam, Nahid Beladi; Ehsani, Marjan; Mortazavi, Hamed; Sabour, Siamak

    2017-01-01

    Introduction The loss of brain cholinergic activity is a key phenomenon in the biochemistry of Alzheimer’s Disease (AD). Due to the specific biosynthesis of Acetylcholinesterase (AChE) of cholinergic neurons, the enzyme has been proposed as a potential biochemical marker of cholinergic activity. AChE is expressed not only in the Central Nervous System (CNS), Peripheral Nervous System (PNS) and muscles, but also on the surface of blood cells and saliva. Aim This study aimed to measure salivary AChE activity in AD and to determine the feasibility of creating a simple laboratory test for diagnosing such patients. Materials and Methods In this cross-sectional study, the recorded data were obtained from 15 Alzheimer’s patients on memantine therapy and 15 healthy subjects. Unstimulated whole saliva samples were collected from the participants and salivary levels of AChE activity were determined by using the Ellman colorimetric method. The Mann Whitney U test was used to compare the average (median) of AChE activity between AD and controls. In order to adjust for possible confounding factors, partial correlation coefficient and multivariate linear regressions were used. Results Although the average of AChE activity in the saliva of people with AD was lower compared to the control group, we found no statistically significant differences using Mann Whitney U test (138 in control group vs. 175 in Alzheimer’s patients, p value=0.25). Additionally, no significant differences were observed in the activity of this enzyme in both sexes or with increased age or duration of the disease. After adjusting for age and gender, there was no association between AChE activity and AD (regression coefficient β=0.08; p value= 0.67). conclusion Saliva AChE activity was not significantly associated with AD. This study might help in introduce a new diagnostic aid for AD or monitor patients with AD. PMID:28274046

  9. Acetylcholinesterase triggers the aggregation of PrP 106-126

    SciTech Connect

    Pera, M.; Roman, S.; Ratia, M.; Camps, P.; Munoz-Torrero, D.; Colombo, L.; Manzoni, C.; Salmona, M.; Badia, A.; Clos, M.V. . E-mail: Victoria.Clos@uab.es

    2006-07-21

    Acetylcholinesterase (AChE), a senile plaque component, promotes amyloid-{beta}-protein (A{beta}) fibril formation in vitro. The presence of prion protein (PrP) in Alzheimer's disease (AD) senile plaques prompted us to assess if AChE could trigger the PrP peptides aggregation as well. Consequently, the efficacy of AChE on the PrP peptide spanning-residues 106-126 aggregation containing a coumarin fluorescence probe (coumarin-PrP 106-126) was studied. Kinetics of coumarin-PrP 106-126 aggregation showed a significant increase of maximum size of aggregates (MSA), which was dependent on AChE concentration. AChE-PrP 106-126 aggregates showed the tinctorial and optical amyloid properties as determined by polarized light and electronic microscopy analysis. A remarkable inhibition of MSA was obtained with propidium iodide, suggesting that AChE triggers PrP 106-126 and A{beta} aggregation through a similar mechanism. Huprines (AChE inhibitors) also significantly decreased MSA induced by AChE as well, unveiling the potential interest for some AChE inhibitors as a novel class of potential anti-prion drugs.

  10. Evaluation of potency of known oximes (pralidoxime, trimedoxime, HI-6, methoxime, obidoxime) to in vitro reactivate acetylcholinesterase inhibited by pesticides (chlorpyrifos and methylchlorpyrifos) and nerve agent (Russian VX).

    PubMed

    Musílek, Kamil; Kuca, Kamil; Jun, Daniel

    2007-01-01

    Nerve agents and pesticides belong to the group of organophosphates. They are able to inhibit irreversibly the enzyme acetylcholinesterase (AChE). Acetylcholinesterase reactivators were designed for the treatment of nerve agent intoxications. Their potency to reactivate pesticide-inhibited AChE was many times evaluated. In this study, five commonly used AChE reactivators (pralidoxime, methoxime, HI-6, obidoxime, trimedoxime) for the reactivation of AChE inhibited by two pesticides (chlorpyrifos and methylchlorpyrifos) were used. Russian VX (nerve agent) as a member of nerve agents' family was taken for comparison. Obtained results show that oximes developed against nerve agent intoxication are less effective for intoxication with organophosphorus pesticides. Especially, methylchlorpyrifos-inhibited AChE was found to be poorly reactivated by the compounds used.

  11. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

    PubMed

    Montenegro, María Fernanda; Cabezas-Herrera, Juan; Campoy, F Javier; Muñoz-Delgado, Encarnación; Vidal, Cecilio J

    2017-02-01

    The observation of acetylcholinesterase (AChE) type H (AChEH), which is the predominant AChE variant in visceral organs and immune cells, in lipid rafts of muscle supports functional reasons for the raft targeting of glypiated AChEH The search for these reasons revealed that liver AChE activity is mostly confined to rafts and that the liver is able to make N-extended AChE variants and target them to rafts. These results prompted us to test whether AChE and muscarinic receptors existed in the same raft. Isolation of flotillin-2-rich raft fractions by their buoyancy in sucrose gradients, followed by immunoadsorption and matrix-assisted laser desorption ionization-time of flight-mass spectrometry application, gave the following results: 1) most hepatic AChE activity emanates from AChE-H mRNA, and its product, glypiated AChEH, accumulates in rafts; 2) N-extended N-AChE readthrough variant, nonglypiated N-AChEH, and N-AChE tailed variant were all identified in liver rafts; and 3) M3 AChRs were observed in rafts, and coprecipitation of raft-confined N-AChE and M3 receptors by using anti-M3 antibodies showed that enzyme and receptor reside in the same raft unit. A raft domain that harbors tightly packed muscarinic receptor and AChE may represent a molecular device that, by means of which, the intensity and duration of cholinergic inputs are regulated.-Montenegro, M. F., Cabezas-Herrera, J., Campoy, F. J., Muñoz-Delgado, E., Vidal, C. J. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

  12. Acetylcholinesterase is associated with apoptosis in β cells and contributes to insulin-dependent diabetes mellitus pathogenesis.

    PubMed

    Zhang, Bao; Yang, Lei; Yu, Luyang; Lin, Bo; Hou, Yanan; Wu, Jun; Huang, Qin; Han, Yifan; Guo, Lihe; Ouyang, Qi; Zhang, Bo; Lu, Lu; Zhang, Xuejun

    2012-03-01

    Acetylcholinesterase (AChE) expression is pivotal during apoptosis. Indeed, AChE inhibitors partially protect cells from apoptosis. Insulin-dependent diabetes mellitus (IDDM) is characterized in part by pancreatic β-cell apoptosis. Here, we investigated the role of AChE in the development of IDDM and analyzed protective effects of AChE inhibitors. Multiple low-dose streptozotocin (MLD-STZ) administration resulted in IDDM in a mouse model. Western blot analysis, cytochemical staining, and immunofluorescence staining were used to detect AChE expression in MIN6 cells, primary β cells, and apoptotic pancreatic β cells of MLD-STZ-treated mice. AChE inhibitors were administered intraperitoneally to the MLD-STZ mice for 30 days. Blood glucose, plasma insulin, and creatine levels were measured, and glucose tolerance tests were performed. The effects of AChE inhibitors on MIN6 cells were also evaluated. AChE expression was induced in the apoptotic MIN6 cells and primary β cells in vitro and pancreatic islets in vivo when treated with STZ. Induction and progressive accumulation of AChE in the pancreatic islets were associated with apoptotic β cells during IDDM development. The administration of AChE inhibitors effectively decreased hyperglycemia and incidence of diabetes, and restored plasma insulin levels and plasma creatine clearance in the MLD-STZ mice. AChE inhibitors partially protected MIN6 cells from the damage caused by STZ treatment. Induction and accumulation of AChE in pancreatic islets and the protective effects of AChE inhibitors on the onset and development of IDDM indicate a close relationship between AChE and IDDM.

  13. Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits*

    PubMed Central

    Ruiz, Carlos A.; Rossi, Susana G.; Rotundo, Richard L.

    2015-01-01

    The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue. PMID:26139603

  14. Comparative study on short- and long-term behavioral consequences of organophosphate exposure: relationship to AChE mRNA expression.

    PubMed

    López-Granero, Caridad; Cardona, Diana; Giménez, Estela; Lozano, Rafael; Barril, José; Aschner, Michael; Sánchez-Santed, Fernando; Cañadas, Fernando

    2014-01-01

    Organophosphates (OPs) affect behavior by inhibiting acetylcholinesterase (AChE). While the cognitive short-term effects may be directly attributed to this inhibition, the mechanisms that underlie OP's long-term cognitive effects remain controversial and poorly understood. Accordingly, two experiments were designed to assess the effects of OPs on cognition, and to ascertain whether both the short- and long-term effects of are AChE-dependent. A single subcutaneous dose of 250 mg/kg chlorpyrifos (CPF), 1.5mg/kg diisopropylphosphorofluoridate (DFP) or 15 mg/kg parathion (PTN) was administered to male Wistar rats. Spatial learning was evaluated 72 h or 23 weeks after exposure, and impulsive choice was tested at 10 and 30 weeks following OPs administration (experiment 1 and 2, respectively). Brain soluble and membrane-bound AChE activity, synaptic AChE-S mRNA, read-through AChE-R mRNA and brain acylpeptide hydrolase (APH) activity (as alternative non-cholinergic target) were analyzed upon completion of the behavioral testing (17 and 37 weeks after OPs exposure). Both short- and long-term CPF treatment caused statistically significant effects on spatial learning, while PTN treatment led only to statistically significant short-term effects. Neither CPF, DFP nor PTN affected the long-term impulsivity response. Long-term exposure to CPF and DFP significantly decreased AChE-S and AChE-R mRNA, while in the PTN treated group only AChE-S mRNA levels were decreased. However, after long-term OP exposure, soluble and membrane-bound AChE activity was indistinguishable from controls. Finally, no changes were noted in brain APH activity in response to OP treatment. Taken together, this study demonstrates long-term effects of OPs on AChE-S and AChE-R mRNA in the absence of changes in AChE soluble and membrane-bound activity. Thus, changes in AChE mRNA expression imply non-catalytic properties of the AChE enzyme.

  15. Inhibition effect of graphene oxide on the catalytic activity of acetylcholinesterase enzyme.

    PubMed

    Wang, Yong; Gu, Yao; Ni, Yongnian; Kokot, Serge

    2015-11-01

    Variations in the enzyme activity of acetylcholinesterase (AChE) in the presence of the nano-material, graphene oxide (GO), were investigated with the use of molecular spectroscopy UV-visible and fluorescence methods. From these studies, important kinetic parameters of the enzyme were extracted; these were the maximum reaction rate, Vm , and the Michaelis constant, Km . A comparison of these parameters indicated that GO inhibited the catalytic activity of the AChE because of the presence of the AChE-GO complex. The formation of this complex was confirmed with the use of fluorescence data, which was resolved with the use of the MCR-ALS chemometrics method. Furthermore, it was found that the resonance light-scattering (RLS) intensity of AChE changed in the presence of GO. On this basis, it was demonstrated that the relationship between AChE and GO was linear and such models were used for quantitative analyses of GO.

  16. Chlorpyrifos and Chlorpyrifos-Oxon Inhibit Axonal Growth by Interfering with the Morphogenic Activity of Acetylcholinesterase

    PubMed Central

    Yang, Dongren; Howard, Angela; Bruun, Donald; Ajua-Alemanj, Mispa; Pickart, Cecile; Lein, Pamela J.

    2008-01-01

    A primary role of acetylcholinesterase (AChE) is regulation of cholinergic neurotransmission by hydrolysis of synaptic acetylcholine. In the developing nervous system, however, AChE also functions as a morphogenic factor to promote axonal growth. This raises the question of whether organophosphorus pesticides (OPs) that are known to selectively bind to and inactivate the enzymatic function of AChE also interfere with its morphogenic function to perturb axonogenesis. To test this hypothesis, we exposed primary cultures of sensory neurons derived from embryonic rat dorsal root ganglia (DRG) to chlorpyrifos (CPF) or its oxon metabolite (CPFO). Both OPs significantly decreased axonal length at concentrations that had no effect on cell viability, protein synthesis or the enzymatic activity of AChE. Comparative analyses of the effects of CPF and CPFO on axonal growth in DRG neurons cultured from AChE nullizygous (AChE−/−) versus wildtype (AChE+/+) mice indicated that while these OPs inhibited axonal growth in AChE+/+ DRG neurons, they had no effect on axonal growth in AChE−/− DRG neurons. However, transfection of AChE−/− DRG neurons with cDNA encoding full-length AChE restored the wildtype response to the axon inhibitory effects of OPs. These data indicate that inhibition of axonal growth by OPs requires AChE, but the mechanism involves inhibition of the morphogenic rather than enzymatic activity of AChE. These findings suggest a novel mechanism for explaining not only the functional deficits observed in children and animals following developmental exposure to OPs, but also the increased vulnerability of the developing nervous system to OPs. PMID:18076960

  17. Acetylcholinesterase of Haematobia irritans (Diptera: Muscidae): Baculovirus expression, biochemical properties and organophosphate insensitivity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirm...

  18. 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...

  19. Biological Synthesis of a Protein Analogue of Acetylcholinesterase: Monoclonal Anti-Idiotype Antibody Analogue of the Esteratic Site

    DTIC Science & Technology

    1987-09-01

    this information to generate oligonucleotide probes in order to screen human cDNA libraries for the presence of the AChE gene. This contract was...this information to generate oligonucleotide probes in order to screen appropriate human cDNA libraries as a first step in the cloning and sequencing...acetylcholinesterase (AChE). The information gleaned from these studies can then be applied to the design and synthesis of chemically or

  20. An optimized extraction technique for acetylcholinesterase inhibitors from the Camellia japonica seed cake by using response surface methodology.

    PubMed

    Kim, Jae Kyeom; Kim, Cho Rong; Lim, Ho-Jeong; Nam, Sang Hae; Joo, Ok Soo; Shin, Dong-Hoon; Shin, Eui-Cheol

    2014-01-01

    The response surface methodology (RSM) was used to optimize the extraction conditions for the acetylcholinesterase (AchE) inhibitory activity and extraction yield from Camellia japonica seed cake. Predicted values for AchE inhibition and extraction yield were 19.41 and 13.35%, respectively, which are in good agreement with the experimental values from validation, suggesting that RSM may provide a useful tool to optimization processes.

  1. Novel acetylcholinesterase target site for malaria mosquito control.

    PubMed

    Pang, Yuan-Ping

    2006-12-20

    Current anticholinesterase pesticides were developed during World War II and are toxic to mammals because they target a catalytic serine residue of acetylcholinesterases (AChEs) in insects and in mammals. A sequence analysis of AChEs from 73 species and a three-dimensional model of a malaria-carrying mosquito (Anopheles gambiae) AChE (AgAChE) reported here show that C286 and R339 of AgAChE are conserved at the opening of the active site of AChEs in 17 invertebrate and four insect species, respectively. Both residues are absent in the active site of AChEs of human, monkey, dog, cat, cattle, rabbit, rat, and mouse. The 17 invertebrates include house mosquito, Japanese encephalitis mosquito, African malaria mosquito, German cockroach, Florida lancelet, rice leaf beetle, African bollworm, beet armyworm, codling moth, diamondback moth, domestic silkworm, honey bee, oat or wheat aphid, the greenbug, melon or cotton aphid, green peach aphid, and English grain aphid. The four insects are house mosquito, Japanese encephalitis mosquito, African malaria mosquito, and German cockroach. The discovery of the two invertebrate-specific residues enables the development of effective and safer pesticides that target the residues present only in mosquito AChEs rather than the ubiquitous serine residue, thus potentially offering an effective control of mosquito-borne malaria. Anti-AgAChE pesticides can be designed to interact with R339 and subsequently covalently bond to C286. Such pesticides would be toxic to mosquitoes but not to mammals.

  2. Imidazole Aldoximes Effective in Assisting Butyrylcholinesterase Catalysis of Organophosphate Detoxification

    PubMed Central

    2015-01-01

    Intoxication by organophosphate (OP) nerve agents and pesticides should be addressed by efficient, quickly deployable countermeasures such as antidotes reactivating acetylcholinesterase or scavenging the parent OP. We present here synthesis and initial in vitro characterization of 14 imidazole aldoximes and their structural refinement into three efficient reactivators of human butyrylcholinesterase (hBChE) inhibited covalently by nerve agent OPs, sarin, cyclosarin, VX, and the OP pesticide metabolite, paraoxon. Rapid reactivation of OP–hBChE conjugates by uncharged and nonprotonated tertiary imidazole aldoximes allows the design of a new OP countermeasure by conversion of hBChE from a stoichiometric to catalytic OP bioscavenger with the prospect of oral bioavailability and central nervous system penetration. The enhanced in vitro reactivation efficacy determined for tertiary imidazole aldoximes compared to that of their quaternary N-methyl imidazolium analogues is attributed to ion pairing of the cationic imidazolium with Asp 70, altering a reactive alignment of the aldoxime with the phosphorus in the OP–hBChE conjugate. PMID:24571195

  3. Imidazole aldoximes effective in assisting butyrylcholinesterase catalysis of organophosphate detoxification.

    PubMed

    Sit, Rakesh K; Fokin, Valery V; Amitai, Gabriel; Sharpless, K Barry; Taylor, Palmer; Radić, Zoran

    2014-02-27

    Intoxication by organophosphate (OP) nerve agents and pesticides should be addressed by efficient, quickly deployable countermeasures such as antidotes reactivating acetylcholinesterase or scavenging the parent OP. We present here synthesis and initial in vitro characterization of 14 imidazole aldoximes and their structural refinement into three efficient reactivators of human butyrylcholinesterase (hBChE) inhibited covalently by nerve agent OPs, sarin, cyclosarin, VX, and the OP pesticide metabolite, paraoxon. Rapid reactivation of OP-hBChE conjugates by uncharged and nonprotonated tertiary imidazole aldoximes allows the design of a new OP countermeasure by conversion of hBChE from a stoichiometric to catalytic OP bioscavenger with the prospect of oral bioavailability and central nervous system penetration. The enhanced in vitro reactivation efficacy determined for tertiary imidazole aldoximes compared to that of their quaternary N-methyl imidazolium analogues is attributed to ion pairing of the cationic imidazolium with Asp 70, altering a reactive alignment of the aldoxime with the phosphorus in the OP-hBChE conjugate.

  4. Development of an in-vivo active reversible butyrylcholinesterase inhibitor

    PubMed Central

    Košak, Urban; Brus, Boris; Knez, Damijan; Šink, Roman; Žakelj, Simon; Trontelj, Jurij; Pišlar, Anja; Šlenc, Jasna; Gobec, Martina; Živin, Marko; Tratnjek, Larisa; Perše, Martina; Sałat, Kinga; Podkowa, Adrian; Filipek, Barbara; Nachon, Florian; Brazzolotto, Xavier; Więckowska, Anna; Malawska, Barbara; Stojan, Jure; Raščan, Irena Mlinarič; Kos, Janko; Coquelle, Nicolas; Colletier, Jacques-Philippe; Gobec, Stanislav

    2016-01-01

    Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD. PMID:28000737

  5. In vitro effect of H2O 2, some transition metals and hydroxyl radical produced via fenton and fenton-like reactions, on the catalytic activity of AChE and the hydrolysis of ACh.

    PubMed

    Méndez-Garrido, Armando; Hernández-Rodríguez, Maricarmen; Zamorano-Ulloa, Rafael; Correa-Basurto, José; Mendieta-Wejebe, Jessica Elena; Ramírez-Rosales, Daniel; Rosales-Hernández, Martha Cecilia

    2014-11-01

    It is well known that the principal biomolecules involved in Alzheimer's disease (AD) are acetylcholinesterase (AChE), acetylcholine (ACh) and the amyloid beta peptide of 42 amino acid residues (Aβ42). ACh plays an important role in human memory and learning, but it is susceptible to hydrolysis by AChE, while the aggregation of Aβ42 forms oligomers and fibrils, which form senile plaques in the brain. The Aβ42 oligomers are able to produce hydrogen peroxide (H2O2), which reacts with metals (Fe(2+), Cu(2+), Cr(3+), Zn(2+), and Cd(2+)) present at high concentrations in the brain of AD patients, generating the hydroxyl radical ((·)OH) via Fenton (FR) and Fenton-like (FLR) reactions. This mechanism generates high levels of free radicals and, hence, oxidative stress, which has been correlated with the generation and progression of AD. Therefore, we have studied in vitro how AChE catalytic activity and ACh levels are affected by the presence of metals (Fe(3+), Cu(2+), Cr(3+), Zn(2+), and Cd(2+)), H2O2 (without Aβ42), and (·) OH radicals produced from FR and FLR. The results showed that the H2O2 and the metals do not modify the AChE catalytic activity, but the (·)OH radical causes a decrease in it. On the other hand, metals, H2O2 and (·)OH radicals, increase the ACh hydrolysis. This finding suggests that when H2O2, the metals and the (·)OH radicals are present, both, the AChE catalytic activity and ACh levels diminish. Furthermore, in the future it may be interesting to study whether these effects are observed when H2O2 is produced directly from Aβ42.

  6. Excess “read-through” acetylcholinesterase attenuates but the “synaptic” variant intensifies neurodeterioration correlates

    PubMed Central

    Sternfeld, Meira; Shoham, Shai; Klein, Omer; Flores-Flores, Cesar; Evron, Tamah; Idelson, Gregory H.; Kitsberg, Dani; Patrick, James W.; Soreq, Hermona

    2000-01-01

    Acute stress increases the risk for neurodegeneration, but the molecular signals regulating the shift from transient stress responses to progressive disease are not yet known. The “read-through” variant of acetylcholinesterase (AChE-R) accumulates in the mammalian brain under acute stress. Therefore, markers of neurodeterioration were examined in transgenic mice overexpressing either AChE-R or the “synaptic” AChE variant, AChE-S. Several observations demonstrate that excess AChE-R attenuates, whereas AChE-S intensifies, neurodeterioration. In the somatosensory cortex, AChE-S transgenics, but not AChE-R or control FVB/N mice, displayed a high density of curled neuronal processes indicative of hyperexcitation. In the hippocampus, AChE-S and control mice, but not AChE-R transgenics, presented progressive accumulation of clustered, heat shock protein 70–immunopositive neuronal fragments and displayed a high incidence of reactive astrocytes. Our findings suggest that AChE-R serves as a modulator that may play a role in preventing the shift from transient, acute stress to progressive neurological disease. PMID:10890884

  7. Honeybee Apis mellifera acetylcholinesterase--a biomarker to detect deltamethrin exposure.

    PubMed

    Badiou, A; Meled, M; Belzunces, L P

    2008-02-01

    The purpose of this study is to investigate the possibility to use acetylcholinesterase (AChE) as a biomarker of exposure to deltamethrin insecticide in the honeybee, Apis mellifera and to test its reliability in the presence of other contaminants, as carbamate insecticide. Joined actions of deltamethrin (pyrethroid) and pirimicarb (carbamate), alone or in association, are investigated on AChE activity in surviving and dead honeybees, with a special focus on the relative proportions of its membrane and soluble forms. At the 0.5X dose (12.5 ng of deltamethrin and/or 2.5 microg of pirimicarb per bee), the residual tissue AChE activity in dead bees was 78% with deltamethrin, 43% with pirimicarb and 33% with dual treatment. In surviving bees, tissue AChE activity represented 250%, and 270% of control AChE activity with deltamethrin and dual treatment, respectively. The analysis of membrane and soluble AChE forms revealed an increase in the soluble form in dead bees after deltamethrin and dual treatment. However, in vitro investigations showed no direct interaction of deltamethrin on soluble and membrane AChE activity. The results suggest that the action of deltamethrin on AChE activity, in honeybee intact organisms, could be due to indirect mechanisms. The duality of AChE response to deltamethrin exposure, exhibited by the possibility of increase (surviving bees) or decrease (dead bees) of its activity has been pointed out for the first time. The important increase in AChE activity in response to deltamethrin, not altered by pirimicarb treatment, suggests that AChE activity could represent a robust biomarker specific to deltamethrin exposure in living bees.

  8. Perspectives for the structure-based design of acetylcholinesterase reactivators.

    PubMed

    Ochoa, Rodrigo; Rodriguez, Carlos A; Zuluaga, Andres F

    2016-07-01

    Rational design of active molecules through structure-based methods has been gaining adepts during the last decades due to the wider availability of protein structures, most of them conjugated with relevant ligands. Acetylcholinesterase (AChE) is a molecular target with a considerable amount of data related to its sequence and 3-dimensional structure. In addition, there are structural insights about the mechanism of action of the natural substrate and drugs used in Alzheimer's disease, organophosphorus compounds, among others. We looked for AChE structural data useful for in silico design of potential interacting molecules. In particular, we focused on information regarding the design of ligands aimed to reactivate AChE catalytic activity. The structures of 178 AChE were annotated and categorized on different subsets according to the nature of the ligand, source organisms and experimental details. We compared sequence homology among the active site from Torpedo californica, Mus musculus and Homo sapiens with the latter two species having the closest relationship (88.9% identity). In addition, the mechanism of organophosphorus binding and the design of effective reactivators are reviewed. A curated data collection obtained with information from several sources was included for researchers working on the field. Finally, a molecular dynamics simulation with human AChE indicated that the catalytic pocket volume stabilizes around 600 Å(3), providing additional clues for drug design.

  9. Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis, widespread in intertropical and temperate regions of the world. Previous cloning, expression, and biochemical characterization of recombinant P. papatasi acetylcholinesterase 1 (PpAChE1) revealed 85% amino acid sequence identity to mosq...

  10. Identical kinetics of human erythrocyte and muscle acetylcholinesterase with respect to carbamate pre-treatment, residual activity upon soman challenge and spontaneous reactivation after withdrawal of the inhibitors.

    PubMed

    Herkert, Nadja M; Eckert, Saskia; Eyer, Peter; Bumm, Rudolf; Weber, Georg; Thiermann, Horst; Worek, Franz

    2008-04-18

    The efficacy of oxime treatment in soman poisoning is limited due to rapid aging of inhibited acetylcholinesterase (AChE). Pre-treatment with carbamates was shown to improve antidotal treatment substantially. Recently, by using a dynamically working in vitro model with real-time determination of membrane-bound AChE activity, we were able to demonstrate that pre-inhibition of human erythrocyte AChE with pyridostigmine or physostigmine resulted in a markedly higher residual AChE activity after inhibition by soman or paraoxon than in the absence of reversible inhibitors. The purpose of the present study was to compare the effect of carbamate pre-treatment and soman challenge with human erythrocyte and muscle homogenate AChE. Both enzyme sources were immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. AChE activity was continuously analyzed in a flow-through detector. Pre-inhibition of AChE with pyridostigmine or physostigmine resulted in a concentration-dependent increase in carbamylation, residual activity after soman inhibition and fraction of decarbamylation AChE after discontinuation of the inhibitors without differences between human erythrocyte and muscle AChE. This data support the view that human erythrocyte AChE is an adequate surrogate marker for synaptic AChE in OP poisoning.

  11. Antisense miR-132 blockade via the AChE-R splice variant mitigates cortical inflammation

    PubMed Central

    Mishra, Nibha; Friedson, Lyndon; Hanin, Geula; Bekenstein, Uriya; Volovich, Meshi; Bennett, Estelle R.; Greenberg, David S.; Soreq, Hermona

    2017-01-01

    MicroRNA (miR)-132 brain-to-body messages suppress inflammation by targeting acetylcholinesterase (AChE), but the target specificity of 3’-AChE splice variants and the signaling pathways involved remain unknown. Using surface plasmon resonance (SPR), we identified preferential miR-132 targeting of soluble AChE-R over synaptic-bound AChE-S, potentiating miR-132-mediated brain and body cholinergic suppression of pro-inflammatory cytokines. Inversely, bacterial lipopolysaccharide (LPS) reduced multiple miR-132 targets, suppressed AChE-S more than AChE-R and elevated inflammatory hallmarks. Furthermore, blockade of peripheral miR-132 by chemically protected AM132 antisense oligonucleotide elevated muscle AChE-R 10-fold over AChE-S, and cortical miRNA-sequencing demonstrated inverse brain changes by AM132 and LPS in immune-related miRs and neurotransmission and cholinergic signaling pathways. In neuromuscular junctions, AM132 co-elevated the nicotinic acetylcholine receptor and AChE, re-balancing neurotransmission and reaching mild muscle incoordination. Our findings demonstrate preferential miR-132-induced modulation of AChE-R which ignites bidirectional brain and body anti-inflammatory regulation, underscoring splice-variant miR-132 specificity as a new complexity level in inflammatory surveillance. PMID:28209997

  12. Preliminary studies of acetylcholinesterase activity in the rat brain using N-phenylferrocenecarboxamide labelled by the technetium-99m.

    PubMed

    Mejri, Najoua; Said, Nadia Malek; Guizani, Sihem; Essouissi, Imen; Saidi, Mouldi

    2013-05-01

    There is currently great interest in developing radiolabeled substrates for acetylcholinesterase that would be useful in the in vivo imaging of patients with Alzheimer's disease. The reduction of acetylcholinesterase (AChE) activity in the brain has been measured in dementia disorders such as Alzheimer's disease and dementia with Lewy bodies using (11)C and (18)F-labeled acetylcholine analogues. Our aim was to develop a new 99mTc-labeled acetylcholine analogue: N-phenylferrocenecarboxamide labelled with technetium-99m (99mTc-TPCC) to study acetylcholinesterase activity. In vivo and in vitro studies demonstrated that the labelled compound was a substrate for acetylcholinesterase. The hydrolytic rate of this substrate was measured and the specificity was evaluated using the inhibitor BW 284 C51. In rat experiments, the 99mTc-TPCC showed desirable properties for studying the acetylcholinesterase in the rat brain: high hydrolytic rate and a moderate specificity of the substrate for acetylcholinesterase.

  13. Acetylcholinesterases from entomopathogenic nematode Heterorhabditid bacteriophora: Susceptibility to insecticides and immunological characteristics.

    PubMed

    Mohamed, Magda A; M E Mahdy, El-Sayed; Ghazy, Abd-El-Hady M; Ibrahim, Nihal M; El-Mezayen, Hatem A; Ghanem, Manal M E

    2017-01-01

    Acetylcholinesterases (AChEs) from the infective juveniles (IJs) of entomopathogenic nematode (EPN) have been investigated with respect to their susceptibility to insecticides and immunological characteristics, aiming at nominating the most compatible insecticide(s) to be used in conjunction with the most insecticide-tolerant EPN strain before incorporation in integrated pest management (IPM) programs. The inhibition kinetics of two purified AChE isoenzymes, AChEAII and AChEBI isolated from Heterorhabditid bacteriophora EM2 strain, by different insecticides revealed that the insensitivity to inhibition by such insecticides could be arranged in a descending order as; methomyl>carbofuran>acetamiprid>oxamyl>malathion. Except for malathion, the insecticides competitively inhibited AChEs with Ki values ranging from 0.1 to 15mM and IC50 values from 1.25 to 23mM. The two AChE isoforms are several folds less sensitive to inhibition by methomyl and carbofuran compared to those previously reported for other insect species. AChEBI was used as an immunogen to raise anti-AChEBI antisera in rabbits. The prepared antisera cross-reacted with AChEs of five different heterorhabditid nematode strains implying the presence of common epitopes shared along all the examined strains. Such studies could aid in the rational selection of the compatible insecticide(s) and the prepared polyclonal anti-AChE antisera would be a valuable immunodiagnostic tool for evaluating the most insecticide-tolerant EPN strain(s) in IPM programs.

  14. Functional analysis and molecular characterization of two acetylcholinesterases from the German cockroach, Blattella germanica.

    PubMed

    Kim, Y H; Choi, J Y; Je, Y H; Koh, Y H; Lee, S H

    2010-12-01

    Two acetylcholinesterases (AChEs; BgAChE1 and BgAChE2) from Blattella germanica were functionally expressed using the baculovirus system. Kinetic analysis demonstrated that BgAChE2 had higher catalytic efficiency but lower substrate specificity than BgAChE1. With the exceptions of paraoxon and propoxur, BgAChE1 was generally less sensitive to inhibitors than BgAChE2. Western blot analysis using anti-BgAChE antibodies revealed that BgAChE1 was far more abundant in all examined tissues compared to BgAChE2, which is only present in the central nervous system. Both BgAChEs existed in dimeric form, covalently connected via a disulphide bridge under native conditions. Most fractions of BgAChE1 had a glycophosphatidylinositol (GPI) anchor, but a small fraction comprised a collagen-like tail. BgAChE2 appeared to have a collagen-GPI-fused tail. Based on the kinetic and molecular properties, tissue distribution and abundance, BgAChE1 was confirmed to play a major role in postsynaptic transmission.

  15. [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.

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

    PubMed

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

    2008-03-01

    The organophosphate insecticide tetrachlorvinphos (TCVP, Rabon) is the active ingredient in "feed-through" larvacides (e.g., Equitrol) for fly control around horse stables. As with other organophosphates, TCVP elicits toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and cholinergic signs. Relatively little is known, however, on the effects of TCVP-containing larvacides on acetylcholinesterase or other esterases in horses. Previous in vitro studies indicated that horse plasma cholinesterase activity was substantially (>10,000-fold) more sensitive than erythrocyte cholinesterase activity to inhibition by TCVP. In the current study, we examined the relative proportion of acetylcholinesterase and butyrylcholinesterase activities in horse plasma and muscle, and evaluated the in vivo effects of Equitrol on target and non-target esterases following oral feeding in horses. In vitro inhibition studies suggested that essentially all cholinesterase activity in horse plasma was butyrylcholinesterase, while muscle contained >90% acetylcholinesterase activity. For in vivo studies, adult, male horses (364-590kg; n=3/treatment group) were given either sweet feed alone or sweet feed supplemented with Equitrol daily for 21 consecutive days at the recommended rate. Clinical signs (vital signs, abdominal auscultation, ophthalmic exam, body temperature) were recorded on a daily basis. Heparinized blood samples were taken at days -1, 1, 3, 7, 21, 28, and 42 while muscle (semimembranosus) biopsies were taken under aseptic conditions on days -1 and 21. No signs of overt toxicity were noted at any time during the study. Plasma cholinesterase activity was significantly inhibited (33%) in larvacide-treated horses as early as one day after treatment and peak inhibition (69-71%) was noted at days 7 and 21. Following cessation of dosing, plasma cholinesterase activity recovered (46% and 83% of control on days 28 and 42, respectively

  17. Selective and Irreversible Inhibitors of Aphid Acetylcholinesterases: Steps Toward Human-Safe Insecticides

    PubMed Central

    Pang, Yuan-Ping; Singh, Sanjay K.; Gao, Yang; Lassiter, T. Leon; Mishra, Rajesh K.; Zhu, Kun Yan; Brimijoin, Stephen

    2009-01-01

    Aphids, among the most destructive insects to world agriculture, are mainly controlled by organophosphate insecticides that disable the catalytic serine residue of acetylcholinesterase (AChE). Because these agents also affect vertebrate AChEs, they are toxic to non-target species including humans and birds. We previously reported that a cysteine residue (Cys), found at the AChE active site in aphids and other insects but not mammals, might serve as a target for insect-selective pesticides. However, aphids have two different AChEs (termed AP and AO), and only AP-AChE carries the unique Cys. The absence of the active-site Cys in AO-AChE might raise concerns about the utility of targeting that residue. Herein we report the development of a methanethiosulfonate-containing small molecule that, at 6.0 µM, irreversibly inhibits 99% of all AChE activity extracted from the greenbug aphid (Schizaphis graminum) without any measurable inhibition of the human AChE. Reactivation studies using β-mercaptoethanol confirm that the irreversible inhibition resulted from the conjugation of the inhibitor to the unique Cys. These results suggest that AO-AChE does not contribute significantly to the overall AChE activity in aphids, thus offering new insight into the relative functional importance of the two insect AChEs. More importantly, by demonstrating that the Cys-targeting inhibitor can abolish AChE activity in aphids, we can conclude that the unique Cys may be a viable target for species-selective agents to control aphids without causing human toxicity and resistance problems. PMID:19194505

  18. Chlorpyrifos and chlorpyrifos-oxon inhibit axonal growth by interfering with the morphogenic activity of acetylcholinesterase

    SciTech Connect

    Yang Dongren; Howard, Angela; Bruun, Donald; Ajua-Alemanj, Mispa; Pickart, Cecile; Lein, Pamela J.

    2008-04-01

    A primary role of acetylcholinesterase (AChE) is regulation of cholinergic neurotransmission by hydrolysis of synaptic acetylcholine. In the developing nervous system, however, AChE also functions as a morphogenic factor to promote axonal growth. This raises the question of whether organophosphorus pesticides (OPs) that are known to selectively bind to and inactivate the enzymatic function of AChE also interfere with its morphogenic function to perturb axonogenesis. To test this hypothesis, we exposed primary cultures of sensory neurons derived from embryonic rat dorsal root ganglia (DRG) to chlorpyrifos (CPF) or its oxon metabolite (CPFO). Both OPs significantly decreased axonal length at concentrations that had no effect on cell viability, protein synthesis or the enzymatic activity of AChE. Comparative analyses of the effects of CPF and CPFO on axonal growth in DRG neurons cultured from AChE nullizygous (AChE{sup -/-}) versus wild type (AChE{sup +/+}) mice indicated that while these OPs inhibited axonal growth in AChE{sup +/+} DRG neurons, they had no effect on axonal growth in AChE{sup -/-} DRG neurons. However, transfection of AChE{sup -/-} DRG neurons with cDNA encoding full-length AChE restored the wild type response to the axon inhibitory effects of OPs. These data indicate that inhibition of axonal growth by OPs requires AChE, but the mechanism involves inhibition of the morphogenic rather than enzymatic activity of AChE. These findings suggest a novel mechanism for explaining not only the functional deficits observed in children and animals following developmental exposure to OPs, but also the increased vulnerability of the developing nervous system to OPs.

  19. Duplication of acetylcholinesterase gene in diamondback moth strains with different sensitivities to acephate.

    PubMed

    Sonoda, Shoji; Shi, Xueyan; Song, Dunlun; Liang, Pei; Gao, Xiwu; Zhang, Youjun; Li, Jianhong; Liu, Yong; Li, Ming; Matsumura, Masaya; Sanada-Morimura, Sachiyo; Minakuchi, Chieka; Tanaka, Toshiharu; Miyata, Tadashi

    2014-05-01

    This study examined the acetylcholinesterase 1 gene (AChE1) in Plutella xylostella strains with different sensitivities to acephate. Multiple haplotypes of the gene were found in the field-collected strains including distinct haplotypes carrying one or both previously reported mutations (A298S and G324A). Moreover, sequencing results indicated the presence of duplicated copies of the gene in the field-collected strains. No correlation was found between copy numbers of AChE1 and levels of resistance to acephate suggesting that extensive AChE1 duplication is not a major resistance factor at least in some P. xylostella strains. Proportions of the A298S and G324A mutations showed no correlation with levels of resistance to acephate. This suggests that acephate resistance of P. xylostella is complex and cannot be evaluated based on the AChE1 copy number or proportions of the resistance mutations alone.

  20. Discovery of Potent Carbonic Anhydrase and Acetylcholinesterase Inhibitors: 2-Aminoindan β-Lactam Derivatives

    PubMed Central

    Genç, Hayriye; Kalin, Ramazan; Köksal, Zeynep; Sadeghian, Nastaran; Kocyigit, Umit M.; Zengin, Mustafa; Gülçin, İlhami; Özdemir, Hasan

    2016-01-01

    β-Lactams are pharmacologically important compounds because of their various biological uses, including antibiotic and so on. β-Lactams were synthesized from benzylidene-inden derivatives and acetoxyacetyl chloride. The inhibitory effect of these compounds was examined for human carbonic anhydrase I and II (hCA I, and II) and acetylcholinesterase (AChE). The results reveal that β-lactams are inhibitors of hCA I, II and AChE. The Ki values of β-lactams (2a–k) were 0.44–6.29 nM against hCA I, 0.93–8.34 nM against hCA II, and 0.25–1.13 nM against AChE. Our findings indicate that β-lactams (2a–k) inhibit both carbonic anhydrases (CA) isoenzymes and AChE at low nanomolar concentrations. PMID:27775608

  1. 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

  2. Reactivation of organophosphate-inhibited human AChE by combinations of obidoxime and HI 6 in vitro.

    PubMed

    Worek, F; Aurbek, N; Thiermann, H

    2007-01-01

    Highly toxic organophosphorus-type (OP) chemical warfare agents (nerve agents) and OP pesticides may be used by terrorists and during military conflicts emphasizing the necessity for the development of effective medical countermeasures. The standard treatment with atropine and acetylcholinesterase (AChE) reactivators (oximes) is considered to be ineffective with certain nerve agents due to low oxime efficacy. Despite research over decades none of the oximes has turned out to be a broad spectrum reactivator to cover the whole range of potential threat agents. The prospective oxime HI 6 is a weak reactivator of tabun- and pesticide-inhibited AChE, while the established oxime obidoxime mainly lacks efficacy with cyclosarin-inhibited enzyme. In order to investigate the feasibility of combining obidoxime and HI 6, human AChE inhibited by sarin, cyclosarin, VX, tabun and paraoxon was reactivated by these oximes either alone or in combination. Two major findings of this study were that a combination of HI 6 and obidoxime did not impair reactivation, compared with HI 6 or obidoxime alone, but broadened the spectrum compared with the individual oximes. By using different oxime concentrations a combination of oxime doses may be suggested which could be an alternative to individual obidoxime or HI 6 autoinjectors.

  3. Salivary Acetylcholinesterase Activity Is Increased in Parkinson's Disease: A Potential Marker of Parasympathetic Dysfunction

    PubMed Central

    Fedorova, Tatyana; Knudsen, Cindy Soendersoe; Mouridsen, Kim; Nexo, Ebba; Borghammer, Per

    2015-01-01

    Introduction. Decreased salivary flow and xerostomia are frequent findings in Parkinson's disease (PD), possibly caused by alterations in the parasympathetic tonus. Here we explore salivary acetylcholinesterase (AChE) activity as a potential biomarker in PD. Methods. We measured salivary flow, AChE activity, and total protein concentration in 30 PD patients and 49 healthy controls. We also performed exploratory correlation analyses with disease duration, motor symptom severity, autonomic complaints, and other nonmotor symptoms. Results. PD patients displayed significantly decreased salivary flow rate, significantly increased salivary AChE activity, and total protein concentration. Importantly, the AChE activity/total protein ratio was significantly increased in PD patients, suggesting that increased AChE activity cannot be explained solely by upconcentration of saliva. The Unified PD Rating Scale (UPDRS) score displayed significant correlation with total salivary protein (P = 0.002) and near-significant correlation with salivary flow (P = 0.07). Color vision test scores were also significantly correlated with AChE activity (P = 0.04) and total protein levels (P = 0.002). Conclusion. Salivary AChE activity is increased in PD patients compared to healthy controls. Future studies are needed to elucidate whether this parameter reflects the extent of neuronal damage and parasympathetic denervation in the salivary glands of PD patients. PMID:25767737

  4. Novel Triazole-Quinoline Derivatives as Selective Dual Binding Site Acetylcholinesterase Inhibitors.

    PubMed

    Mantoani, Susimaire P; Chierrito, Talita P C; Vilela, Adriana F L; Cardoso, Carmen L; Martínez, Ana; Carvalho, Ivone

    2016-02-05

    Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder worldwide. Currently, the only strategy for palliative treatment of AD is to inhibit acetylcholinesterase (AChE) in order to increase the concentration of acetylcholine in the synaptic cleft. Evidence indicates that AChE also interacts with the β-amyloid (Aβ) protein, acting as a chaperone and increasing the number and neurotoxicity of Aβ fibrils. It is known that AChE has two binding sites: the peripheral site, responsible for the interactions with Aβ, and the catalytic site, related with acetylcholine hydrolysis. In this work, we reported the synthesis and biological evaluation of a library of new tacrine-donepezil hybrids, as a potential dual binding site AChE inhibitor, containing a triazole-quinoline system. The synthesis of hybrids was performed in four steps using the click chemistry strategy. These compounds were evaluated as hAChE and hBChE inhibitors, and some derivatives showed IC50 values in the micro-molar range and were remarkably selective towards hAChE. Kinetic assays and molecular modeling studies confirm that these compounds block both catalytic and peripheral AChE sites. These results are quite interesting since the triazole-quinoline system is a new structural scaffold for AChE inhibitors. Furthermore, the synthetic approach is very efficient for the preparation of target compounds, allowing a further fruitful new chemical library optimization.

  5. Synaptosomal acetylcholinesterase activity variation pattern in the presence of electromagnetic fields.

    PubMed

    Afrasiabi, Ali; Riazi, Gholam Hossein; Abbasi, Shayan; Dadras, Ali; Ghalandari, Behafarid; Seidkhani, Hossein; Modaresi, Seyed Mohamad Sadegh; Masoudian, Neda; Amani, Amir; Ahmadian, Shahin

    2014-04-01

    Acetylcholinesterase (AChE) is the enzyme that controls the acetylcholine (ACh) concentrations in cholinergic synaptic clefts by hydrolyzing ACh to choline and acetate. Cholinergic synapses are involved in important functions such as learning, memory and cognition. In this study, we investigated the effects of a wide range of extremely low frequency electromagnetic fields (ELF-EMFs) on synaptic ACh concentrations through AChE enzyme activity assay. Synaptosome suspensions were prepared as a neural terminus from cerebral cortex of sheep brain. Prepared synaptosomes were exposed to ELF-EMFs with frequency ranging from 50 Hz to 230 Hz for duration between 15 and 120 min and flux intensity between 0.1 mT and 1.7 mT. Consequently, AChE activity was measured by Ellman method. Raw data were analyzed by neural network based software, Inform 4.02, to predict AChE activity pattern through nine 3D curves. These curves showed that AChE activity decreases when exposed to ELF-EMFs of 1.2 mT to 1.7 mT intensity and 50 Hz to 90 Hz frequency. Thus, it is proposed that exposure to fields of in this range of frequency-intensity would be effective in clinical treatments of cholinergic disorders to increase synaptic ACh concentration. However, more in vivo experiments are needed to develop this suggested treatment.

  6. Absence of substrate inhibition and freezing-inactivation of the mosquito acetylcholinesterase are caused by alterations of hydrophobic interactions.

    PubMed

    Dary, O; Wedding, R T

    1990-05-31

    Membrane-bound acetylcholinesterase (AChE) from mosquito showed the characteristic substrate inhibition of this enzyme, but 105,000 x g supernatants of freshly extracted enzyme did not. Addition of chaotropic anions, a freeze-thaw cycle and autolysis of the amphiphilic acetylcholinesterase to its non-amphiphilic derivatives resulted in return of the substrate inhibition feature along with an apparent increment in the enzyme activity. These results suggested that the lipidic environment of the mosquito AChE is temporarily perturbed when extracted. The enzyme is probably trapped in non-sedimenting mixtures composed of endogenous amphiphilic molecules. The occurrence of this phenomenon was not affected by the presence of Triton X-100 and other detergents, either alone or in combination with sodium chloride. Freezing in the presence of strong chaotropic anions (perchlorate, iodide and thiocyanate) caused the irreversible inactivation of the mosquito AChE. Crude and incomplete purified fractions of the enzyme were more sensitive than a more purified preparation. With both the purified AChE and the non-purified AChE, amphiphilic AChE was more freeze labile. Freezing at -10 degrees C enhanced inactivation of non-purified fractions. At this temperature, even weak chaotropic anions (fluoride, chloride and nitrate), while in combination with non-ionic detergents that solubilized mosquito AChE efficiently, reduced the enzyme activity of these fractions. In this case, recovery of the enzyme activity by incubation at 25 degrees C was inversely correlated with the effectiveness of the chaotropic anion. Gel filtration failed to show any change in the hydrodynamic radius of the freezing-inactivated AChE. Therefore, this phenomenon is explained as different degrees of denaturation of the enzyme in direct association with the chaotropic strength. Thus, antichaotropic anions, such as sulfate, should improve the stability of the mosquito acetylcholinesterase during extraction

  7. Acetylcholinesterase Inhibitors with Photoswitchable Inhibition of β-Amyloid Aggregation

    PubMed Central

    2014-01-01

    Photochromic cholinesterase inhibitors were obtained from cis-1,2-α-dithienylethene-based compounds by incorporating one or two aminopolymethylene tacrine groups. All target compounds are potent acetyl- (AChE) and butyrylcholinesterase (BChE) inhibitors in the nanomolar concentration range. Compound 11b bearing an octylene linker exhibited interactions with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Yet upon irradiation with light, the mechanism of interaction varied from one photochromic form to another, which was investigated by kinetic studies and proved “photoswitchable”. The AChE-induced β-amyloid (Aβ) aggregation assay gave further experimental support to this finding: Aβ1–40 aggregation catalyzed by the PAS of AChE might be inhibited by compound 11b in a concentration-dependent manner and seems to occur only with one photochromic form. Computational docking studies provided potential binding modes of the compound. Docking studies and molecular dynamics (MD) simulations for the ring-open and -closed form indicate a difference in binding. Although both forms can interact with the PAS, more stable interactions are observed for the ring-open form based upon stabilization of a water molecule network within the enzyme, whereas the ring-closed form lacks the required conformational flexibility for an analogous binding mode. The photoswitchable inhibitor identified might serve as valuable molecular tool to investigate the different biological properties of AChE as well as its role in pathogenesis of AD in in vitro assays. PMID:24628027

  8. Huperzia quadrifariata and Huperzia reflexa alkaloids inhibit acetylcholinesterase activity in vivo in mice brain.

    PubMed

    Konrath, E L; Neves, B M; Passos, C Dos S; Lunardi, P S; Ortega, M G; Cabrera, J L; Gonçalves, C A; Henriques, A T

    2012-11-15

    Huperzine A, a Lycopodium alkaloid produced by Chinese folk herb Huperzia serrata (Lycopodiaceae), has been shown to be a promising agent for the treatment of Alzheimer's disease due to its potent acetylcholinesterase (AChE) activity, as well its efficacy in the treatment of memory of aged patients. Thus, the effects of two Huperzia species of habitats in Brazil (H. quadrifariata and H. reflexa) with described in vitro AChE inhibition activities were studied and their effects on mice brain AChE inhibition were determined after a single intraperitoneal (i.p.) injection. The alkaloid extracts were administered to mice in various doses (10, 1 and 0.5mg/kg) and acetylcholinesterase activity was measured post mortem in two brain areas using the Ellman's colorimetric method. The AChE activity was found to be significantly reduced in both the cortex and hippocampus, although this activity was less potent than that of reference inhibitor huperzine A (0.5mg/kg). Thus, it appears that H. quadrifariata and H. reflexa alkaloid extracts, shown to inhibit acetylcholinesterase in vitro, also have very potent in vivo effects, suggesting that the Huperzia species may still constitute a promising source of compounds with pharmaceutical interest for Alzheimer's disease.

  9. Experimental and computational studies on the inhibition of acetylcholinesterase by curcumin and some of its derivatives.

    PubMed

    Tello-Franco, Veronica; Lozada-García, Maria Concepcion; Soriano-García, Manuel

    2013-06-01

    Recent studies have demonstrated several biological activities of curcumin with therapeutic potential against Alzheimer's disease, among them the inhibition of the enzyme acetylcholinesterase (AChE). Aiming at identifying the chemical features relevant for this activity, the inhibition of curcumin and a set of 7 derivatives against AChE of E. electricus was measured. These derivatives presented lower activity than curcumin, allowing for the identification of possible unfavorable enzyme-inhibitor interactions. Our computational approach was to dock the molecules to the active site of AChE, followed by an analysis of hydrogen bonds and close contacts to relevant aromatic amino acid residues. To account for inhibitory activity, we sought to define the common structural features between known acetylcholinesterase inhibitors and the tested derivatives. A pharmacophore model was generated, which consisted of two hydrophobic, one aromatic and one hydrogen bond acceptor features. We conclude that the presence of two aromatic rings and the distance between them, allows curcumin and its derivatives to favorably interact with both the quaternary and peripheral sites of AChE. Hydrogen bonds can be formed with the quaternary and acyl sites, which should further stabilize the complex. The acylation of the hydroxyl groups and the reduction of the conjugated double bonds lowered the inhibitory activity, pointing to the modification of the keto-enol moiety as the best alternative for the design of more potent curcumin derivatives as acetylcholinesterase inhibitors.

  10. A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens.

    PubMed

    Malcolm, C A; Bourguet, D; Ascolillo, A; Rooker, S J; Garvey, C F; Hall, L M; Pasteur, N; Raymond, M

    1998-05-01

    An acetylcholinesterase (AChE) gene, Ace.x, showing 93% identity of deduced amino acid sequence to Anopheles stephensi Ace has been cloned from a Culex pipiens strain homozygous for insensitive AChE (iAChE) mediated insecticide resistance. DNA sequence of genomic DNA clones identified exons 2-5. RFLP of six clones indicated four possible alleles. Linkage analysis located Ace.x to chromosome I, less than 0.8 centimorgans from the sex locus, whereas the locus conferring resistance was 2.0 centimorgans from plum-eye on chromosome II. Ace.1 coding for AChE1, which is associated with resistance, is therefore autosomal. We propose that Ace.x is the recently postulated Ace.2 coding for the biochemically distinct AChE2, which is not associated with resistance.

  11. Three N-Glycosylation Sites of Human Acetylcholinesterase Shares Similar Glycan Composition.

    PubMed

    Xu, Miranda L; Luk, Wilson K W; Lau, Kei M; Bi, Cathy W C; Cheng, Anthony W M; Gong, Amy G W; Lin, Huangquan; Tsim, Karl W K

    2015-12-01

    Acetylcholinesterase (AChE; EC 3.1.1.7) is a glycoprotein possessing three conserved N-linked glycosylation sites in mammalian species, locating at 296, 381, and 495 residues of the human sequence. Several lines of evidence demonstrated that N-glycosylation of AChE affected the enzymatic activity, as well as its biosynthesis. In order to determine the role of three N-glycosylation sites in AChE activity and glycan composition, the site-directed mutagenesis of N-glycosylation sites in wild-type human AChE(T) sequence was employed to generate the single-site mutants (i.e., AChE(T) (N296Q), AChET (N381Q), and AChE(T) (N495Q)) and all site mutant (i.e., AChE(T) (3N→3Q)). The mutation did not affect AChE protein expression in the transfected cells. The mutants, AChE(T) (3N→3Q) and AChE(T) (N381Q), showed very minimal enzymatic activity, while the other mutants showed reduced activity. By binding to lectins, Con A, and SNA, the glycosylation profile was revealed in those mutated AChE. The binding affinity with lectins showed no significant difference between various N-glycosylation mutants, which suggested that similar glycan composition should be resulted from different N-glycosylation sites. Although the three glycosylation sites within AChE sequence have different extent in affecting the enzymatic activity, their glycan compositions are very similar.

  12. Biochemical and toxicological properties of two acetylcholinesterases from the common bed bug, Cimex lectularius.

    PubMed

    Hwang, Chae Eun; Kim, Young Ho; Kwon, Deok Ho; Seong, Keon Mook; Choi, Jae Young; Je, Yeon Ho; Lee, Si Hyeock

    2014-03-01

    We examined the molecular and enzymatic properties of two acetylcholinesterases (AChEs; ClAChE1 and ClAChE2) from the common bed bug, Cimex lectularius. Native polyacrylamide gel electrophoresis followed by activity staining and Western blotting revealed that ClAChE1 is the main catalytic enzyme and is abundantly expressed in various tissues. Both ClAChEs existed in dimeric form connected by a disulfide bridge and were attached to the membrane via a glycophosphatidylinositol anchor. To determine their kinetic and inhibitory properties, both ClAChE1 and ClAChE2 were in vitro expressed in Sf9 cells using a baculovirus expression system. ClAChE1 showed higher catalytic efficiency toward acetylcholine, supporting the hypothesis that ClAChE1 plays a major role in postsynaptic transmission. An inhibition assay revealed that ClAChE1 is generally more sensitive to organophosphates and carbamates examined although ClAChE2 was >4000-fold more sensitive to malaoxon than ClAChE1. The relatively higher correlation between the in vitro ClAChE1 inhibition and the in vivo toxicity suggested that ClAChE1 is the more relevant toxicological target for organophosphates and carbamates. Although the physiological function of ClAChE2 remains to be elucidated, ClAChE2 also appears to have neuronal functions, as judged by its tissue distribution and molecular and kinetic properties. Our findings help expand our knowledge on insect AChEs and their toxicological properties.

  13. Acetylcholinesterase Activity and Neurodevelopment in Boys and Girls

    PubMed Central

    Himes, John H.; Jacobs, David R.; Alexander, Bruce H.; Gunnar, Megan R.

    2013-01-01

    BACKGROUND: Organophosphate exposures can affect children’s neurodevelopment, possibly due to neurotoxicity induced by acetylcholinesterase (AChE) inhibition, and may affect boys more than girls. We tested the hypothesis that lower AChE activity is associated with lower neurobehavioral development among children living in Ecuadorian floricultural communities. METHODS: In 2008, we examined 307 children (age: 4–9 years; 52% male) and quantified AChE activity and neurodevelopment in 5 domains: attention/executive functioning, language, memory/learning, visuospatial processing, and sensorimotor (NEPSY-II test). Associations were adjusted for demographic and socioeconomic characteristics and height-for-age, flower worker cohabitation, and hemoglobin concentration. RESULTS: Mean ± standard deviation AChE activity was 3.14 ± 0.49 U/mL (similar for both genders). The range of scores among neurodevelopment subtests was 5.9 to 10.7 U (standard deviation: 2.6–4.9 U). Girls had a greater mean attention/executive functioning domain score than boys. In boys only, there were increased odds ratios of low (<9th percentile) neurodevelopment among those in the lowest tertile versus the highest tertile of AChE activity (odds ratios: total neurodevelopment: 5.14 [95% confidence interval (CI): 0.84 to 31.48]; attention/executive functioning domain: 4.55 [95% CI: 1.19 to 17.38], memory/learning domain: 6.03 [95% CI: 1.17 to 31.05]) after adjustment for socioeconomic and demographic factors, height-for-age, and hemoglobin. Within these domains, attention, inhibition and long-term memory subtests were most affected. CONCLUSIONS: Low AChE activity was associated with deficits in neurodevelopment, particularly in attention, inhibition, and memory in boys but not in girls. These critical cognitive skills affect learning and academic performance. Added precautions regarding secondary occupational pesticide exposure would be prudent. PMID:24249815

  14. Landscape and pesticide effects on honey bees: forager survival and expression of acetylcholinesterase and brain oxidative genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of the present work was to assess the effects of agricultural pesticides on honey bee (Apis mellifera L.) survival and physiological stress. Integrated use of acetylcholinesterase (AChE) and antioxidant enzymes (catalase and glutathione S-transferase) was tested on honey bee brains for detec...

  15. Inhibitor Profile of bis(n)-tacrines and N-methylcarbamates on Acetylcholinesterase from Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAchE) compared to h...

  16. Acetylcholinesterase 1 in populations of organophosphate resistant North American strains of the cattle tick, Rhipicephalus microplus (Acari: Ixodidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a collaboration with Purdue University researchers, we sequenced a 143,606 base pair Rhipicephalus microplus BAC library clone that contained the coding region for acetylcholinesterase 1 (AChE1). Sequencing was by Sanger protocols and the final assembly resulted in 15 contigs of varying length, e...

  17. Intensified vmPFC surveillance over PTSS under perturbed microRNA-608/AChE interaction.

    PubMed

    Lin, T; Simchovitz, A; Shenhar-Tsarfaty, S; Vaisvaser, S; Admon, R; Hanin, G; Hanan, M; Kliper, E; Bar-Haim, Y; Shomron, N; Fernandez, G; Lubin, G; Fruchter, E; Hendler, T; Soreq, H

    2016-05-03

    Trauma causes variable risk of posttraumatic stress symptoms (PTSS) owing to yet-unknown genome-neuronal interactions. Here, we report co-intensified amygdala and ventromedial prefrontal cortex (vmPFC) emotional responses that may overcome PTSS in individuals with the single-nucleotide polymorphism (SNP) rs17228616 in the acetylcholinesterase (AChE) gene. We have recently shown that in individuals with the minor rs17228616 allele, this SNP interrupts AChE suppression by microRNA (miRNA)-608, leading to cortical elevation of brain AChE and reduced cortisol and the miRNA-608 target GABAergic modulator CDC42, all stress-associated. To examine whether this SNP has effects on PTSS and threat-related brain circuits, we exposed 76 healthy Israel Defense Forces soldiers who experienced chronic military stress to a functional magnetic resonance imaging task of emotional and neutral visual stimuli. Minor allele individuals predictably reacted to emotional stimuli by hyperactivated amygdala, a hallmark of PTSS and a predisposing factor of posttraumatic stress disorder (PTSD). Despite this, minor allele individuals showed no difference in PTSS levels. Mediation analyses indicated that the potentiated amygdala reactivity in minor allele soldiers promoted enhanced vmPFC recruitment that was associated with their limited PTSS. Furthermore, we found interrelated expression levels of several miRNA-608 targets including CD44, CDC42 and interleukin 6 in human amygdala samples (N=7). Our findings suggest that miRNA-608/AChE interaction is involved in the threat circuitry and PTSS and support a model where greater vmPFC regulatory activity compensates for amygdala hyperactivation in minor allele individuals to neutralize their PTSS susceptibility.

  18. Intensified vmPFC surveillance over PTSS under perturbed microRNA-608/AChE interaction

    PubMed Central

    Lin, T; Simchovitz, A; Shenhar-Tsarfaty, S; Vaisvaser, S; Admon, R; Hanin, G; Hanan, M; Kliper, E; Bar-Haim, Y; Shomron, N; Fernandez, G; Lubin, G; Fruchter, E; Hendler, T; Soreq, H

    2016-01-01

    Trauma causes variable risk of posttraumatic stress symptoms (PTSS) owing to yet-unknown genome–neuronal interactions. Here, we report co-intensified amygdala and ventromedial prefrontal cortex (vmPFC) emotional responses that may overcome PTSS in individuals with the single-nucleotide polymorphism (SNP) rs17228616 in the acetylcholinesterase (AChE) gene. We have recently shown that in individuals with the minor rs17228616 allele, this SNP interrupts AChE suppression by microRNA (miRNA)-608, leading to cortical elevation of brain AChE and reduced cortisol and the miRNA-608 target GABAergic modulator CDC42, all stress-associated. To examine whether this SNP has effects on PTSS and threat-related brain circuits, we exposed 76 healthy Israel Defense Forces soldiers who experienced chronic military stress to a functional magnetic resonance imaging task of emotional and neutral visual stimuli. Minor allele individuals predictably reacted to emotional stimuli by hyperactivated amygdala, a hallmark of PTSS and a predisposing factor of posttraumatic stress disorder (PTSD). Despite this, minor allele individuals showed no difference in PTSS levels. Mediation analyses indicated that the potentiated amygdala reactivity in minor allele soldiers promoted enhanced vmPFC recruitment that was associated with their limited PTSS. Furthermore, we found interrelated expression levels of several miRNA-608 targets including CD44, CDC42 and interleukin 6 in human amygdala samples (N=7). Our findings suggest that miRNA-608/AChE interaction is involved in the threat circuitry and PTSS and support a model where greater vmPFC regulatory activity compensates for amygdala hyperactivation in minor allele individuals to neutralize their PTSS susceptibility. PMID:27138800

  19. Microcalorimetric study of the inhibition of butyrylcholinesterase by paraoxon.

    PubMed

    Debord, Jean; Harel, Michel; Verneuil, Bernard; Bollinger, Jean-Claude; Dantoine, Thierry

    2009-06-15

    The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by the organophosphorus compound paraoxon (diethyl 4-nitrophenyl phosphate) was studied by flow microcalorimetry at 37 degrees C in Tris buffer (pH 7.5) using a modification of the kinetic model described by Stojan and coworkers [J. Stojan, V. Marcel, S. Estrada-Mondaca, A. Klaebe, P. Masson, D. Fournier, A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase, FEBS Lett. 440 (1998) 85-88]. The reversible steps of the inhibition were studied in the mixing cell of the calorimeter, whereas the irreversible step was studied in the flow-through cell. A new pseudo-first-order approximation was developed to allow the kinetic analysis of inhibition progress curves in the presence of substrate when a significant amount of substrate is transformed. This approximation also allowed one to compute an analytical expression of the calorimetric curves using a gamma distribution to describe the impulse response of the calorimeter. Fitting models to data by nonlinear regression, with simulated annealing as a stochastic optimization method, allowed the determination of all kinetic parameters. It was found that paraoxon binds to both the enzyme and acyl-enzyme, but with weak affinities (K(i) = 0.123 mM and K'(i) = 5.5 mM). A slight activation was observed at the lowest paraoxon concentrations and was attributed to the binding of the substrate to the enzyme-inhibitor complex. The bimolecular inhibition rate constant k(i) = 2.8 x 10(4) M(-1) s(-1) was in agreement with previous studies. It is hoped that the methods developed in this work will contribute to extending the application range of microcalorimetry in the field of irreversible inhibitors.

  20. Production of Enzymatically Active Human Acetylcholinesterase in E. Coli

    DTIC Science & Technology

    1993-10-01

    AD-A282 703 lE1l1lm11I AD( CONTRACT NO: DAMD17-90-C-0107 TITLE: PRODUCTION OF ENZYMATICALLY ACTIVE HUMAN ACETYLCHOLINESTERASE IN E . COLI PRINCIPAL...FUNDING NUMBERS Production of Enzymatically Active Human Contract No. Acetylcholinesterase in E . coli DAMD17-90-C-0107 6. AUTHOR(S) M. Gorecki, Ph.D. and M...S493pMFL-52Ser - Run #1 37 Table 8: Summary of reconstitution and purification of rhAChE derived from E . coli S493pMFL-52Ser - Run #2 38 Table 9

  1. Reactivation steps by 2-PAM of tabun-inhibited human acetylcholinesterase: reducing the computational cost in hybrid QM/MM methods.

    PubMed

    da Silva Gonçalves, Arlan; França, Tanos Celmar Costa; Caetano, Melissa Soares; Ramalho, Teodorico Castro

    2014-01-01

    The present work describes a simple integrated Quantum Mechanics/Molecular Mechanics method developed to study the reactivation steps by pralidoxime (2-PAM) of acetylcholinesterase (AChE) inhibited by the neurotoxic agent Tabun. The method was tested on an AChE model and showed to be able to corroborate most of the results obtained before, through a more complex and time-consuming methodology, proving to be suitable to this kind of mechanistic study at a lower computational cost.

  2. Different inhibition of acetylcholinesterase in selected parts of the rat brain following intoxication with VX and Russian VX.

    PubMed

    Hajek, Petr; Bajgar, Jiri; Slizova, Dasa; Krs, Otakar; Kuca, Kamil; Capek, Lukas; Fusek, Josef

    2009-01-01

    Differences between acetylcholinesterase (AChE) inhibition in the brain structures following VX and RVX exposure are not known as well as information on the possible correlation of biochemical and histochemical methods detecting AChE activity. Therefore, inhibition of AChE in different brain parts detected by histochemical and biochemical techniques was compared in rats intoxicated with VX and RVX. AChE activities in defined brain regions 30 min after treating rats with VX and Russian VX intramuscularly (1.0 x LD(50)) were determined by using biochemical and histochemical methods. AChE inhibition was less expressed for RVX, in comparison with VX. Frontal cortex and pontomedullar areas containing ncl. reticularis has been found as the most sensitive areas for the action of VX. For RVX, these structures were determined to be frontal cortex, dorsal septum, and hippocampus, respectively. Histochemical and biochemical results were in good correlation (R(xy) = 0.8337). Determination of AChE activity in defined brain structures was a more sensitive parameter for VX or RVX exposure than the determination of AChE activity in the whole-brain homogenate. This activity represents a "mean" of the activities in different structures. Thus, AChE activity is the main parameter investigated in studies searching for target sites following nerve-agent poisoning contributing to better understanding of toxicodynamics of nerve agents.

  3. Identification and Molecular Characterization of Two Acetylcholinesterases from the Salmon Louse, Lepeophtheirus salmonis

    PubMed Central

    Kaur, Kiranpreet; Bakke, Marit Jørgensen; Nilsen, Frank; Horsberg, Tor Einar

    2015-01-01

    Acetylcholinesterase (AChE) is an important enzyme in cholinergic synapses. Most arthropods have two genes (ace1 and ace2), but only one encodes the predominant synaptic AChE, the main target for organophosphates. Resistance towards organophosphates is widespread in the marine arthropod Lepeophtheirus salmonis. To understand this trait, it is essential to characterize the gene(s) coding for AChE(s). The full length cDNA sequences encoding two AChEs in L. salmonis were molecularly characterized in this study. The two ace genes were highly similar (83.5% similarity at protein level). Alignment to the L. salmonis genome revealed that both genes were located close to each other (separated by just 26.4 kbp on the L. salmonis genome), resulting from a recent gene duplication. Both proteins had all the typical features of functional AChE and clustered together with AChE-type 1 proteins in other species, an observation that has not been described in other arthropods. We therefore concluded the presence of two versions of ace1 gene in L. salmonis, named ace1a and ace1b. Ace1a was predominantly expressed in different developmental stages compared to ace1b and was possibly active in the cephalothorax, indicating that ace1a is more likely to play the major role in cholinergic synaptic transmission. The study is essential to understand the role of AChEs in resistance against organophosphates in L. salmonis. PMID:25938836

  4. Carbonic anhydrase and acetylcholinesterase inhibitory effects of carbamates and sulfamoylcarbamates.

    PubMed

    Göçer, Hülya; Akincioğlu, Akın; Göksu, Süleyman; Gülçin, İlhami; Supuran, Claudiu T

    2015-04-01

    Carbonic anhydrases (CA), as a family of metalloenzymes, are found in almost every type of tissue and play an important role in catalyzing the equilibration of carbon dioxide and carbonic acid. In this study, a series of carbamate derivative was synthesized, and their inhibition effects on hCA I, hCA II and acetylcholinesterase (AChE) enzymes were investigated. They were determined to be very good inhibitor against for both isoenzymes (hCA I and hCA II) and AChE. The hCA I and hCA II were effectively inhibited by the carbamate derivatives, with inhibition constants (Ki) in the range of 194.4-893.5 nM (for hCA I) and 103.9-835.7 nM (for hCA II). On the other hand, Ki parameters of these compounds for AChE enzyme inhibition were determined in the range of 12.0-61.3 nM. The results clearly showed that both CA isoenzymes and AChE were inhibited by carbamate derivatives at the nM levels.

  5. Kinetic analysis of interactions of different sarin and tabun analogues with human acetylcholinesterase and oximes: is there a structure-activity relationship?

    PubMed

    Aurbek, Nadine; Herkert, Nadja M; Koller, Marianne; Thiermann, Horst; Worek, Franz

    2010-09-06

    The repeated misuse of highly toxic organophosphorus compound (OP) based chemical warfare agents in military conflicts and terrorist attacks poses a continuous threat to the military and civilian sector. The toxic symptomatology of OP poisoning is mainly caused by inhibition of acetylcholinesterase (AChE, E.C. 3.1.1.7) resulting in generalized cholinergic crisis due to accumulation of the neurotransmitter acetylcholine (ACh) in synaptic clefts. Beside atropine as competitive antagonist of ACh at muscarinic ACh receptors oximes as reactivators of OP-inhibited AChE are a mainstay of standard antidotal treatment. However, human AChE inhibited by certain OP is rather resistant to oxime-induced reactivation. The development of more effective oxime-based reactivators may fill the gaps. To get more insight into a potential structure-activity relationship between human AChE, OPs and oximes in vitro studies were conducted to investigate interactions of different tabun and sarin analogues with human AChE and the oximes obidoxime and HI 6 by determination of various kinetic constants. Rate constants for the inhibition of human AChE by OPs, spontaneous dealkylation and reactivation as well as reactivation by obidoxime and HI 6 of OP-inhibited human AChE were determined. The recorded kinetic data did not allow a general statement concerning a structure-activity relationship between human AChE, OP and oximes.

  6. Carbamate and Organophosphorus Nematicides: Acetylcholinesterase inhibition and Effects on Dispersal.

    PubMed

    Pree, D J; Townshend, J L; Archibald, D E

    1989-10-01

    The sensitivities of acetylcholinesterases (ACHE) from the fungus-feeder Aphelenchus avenae and the plant-parasitic species Helicotylenchus dihystera and Pratylenchus penetrans and the housefly, Musca domestica, were compared using a radiometric assay which utilized H(3) acetylcholine as a substrate. Nematode ACHE were generally less sensitive to inhibition by organophosphorns and carbamate pesticides than were ACHE from the housefly. ACHE from the plant-parasitic species and A. avenae were generally similar in sensitivity. In soil, carbamates were more toxic than the organophosphorus pesticides to A. avenae. All pesticides tested affected nematode movement, but fenamiphos was more inhibitory than others. The effects on dispersal of nematodes may be an important mechanism in control by some nematicides.

  7. Acetylcholinesterase-inhibitory activities of the extracts from sponges collected in mauritius waters.

    PubMed

    Beedessee, Girish; Ramanjooloo, Avin; Surnam-Boodhun, Rashmee; van Soest, Rob W M; Marie, Daniel E P

    2013-03-01

    Patients diagnosed with Alzheimer's disease (AD) show a characteristic neurochemical deficit of acetylcholine, especially in the basal forebrains. The use of acetylcholinesterase (AChE) inhibitors to retard the hydrolysis of acetylcholine has been suggested as a promising strategy for AD treatment. In this study, we evaluated the acetylcholinesterase inhibitory (AChEI) activities of 134 extracts obtained from 45 species of marine sponges. Thin-layer chromatography (TLC) and microplate assays reveal potent acetylcholinsterase inhibitory activities of two AcOEt extracts from the sponges Pericharax heteroraphis and Amphimedon navalis PULITZER-FINALI. We further investigated the inhibitory kinetics of the extracts and found them to display mixed competitive/noncompetitive inhibition and associated their inhibitory activity partly to terpenoids. Acetylcholinesterase inhibitors from marine organisms have been rarely studied, and this study demonstrated the potential of marine sponges as a source of pharmaceutical leads against neurodegenerative diseases.

  8. Cholinesterase Inhibitor Therapy in Alzheimer’s: The limits and tolerability of Irreversible CNS-selective Acetylcholinesterase Inhibition in Primates

    PubMed Central

    Moss, Donald E.; Perez, Ruth G.; Kobayashi, Haruo

    2016-01-01

    Irreversible acetylcholinesterase (AChE) inhibition accumulates to high levels in the central nervous system (CNS) because AChE turnover in the brain is much slower than in peripheral tissues. As expected from this CNS selectivity, the irreversible AChE inhibitor methanesulfonyl fluoride (MSF) produces significant cognitive improvement in Alzheimer’s patients without the gastrointestinal toxicity that plagues other AChE inhibitors. However, without dose-limiting gastrointestinal toxicity, one shortcoming of the prior human studies of MSF is that the upper limits of CNS AChE inhibition that might be tolerated could not be tested. Therefore, in this study, monkeys were treated with escalating intramuscular doses of MSF that culminated with several weeks of 1.5 mg/kg dosing, more than eight times the prior human clinical dose, still without signs of toxicity. Brain biopsies showed that ~ 80% AChE inhibition had been produced and that the new synthesis of cortical AChE had a half-time (t1/2) of ~ 12 days. A single IM dose of 1.5 mg/kg MSF produced ~ 59% inhibition in cerebrospinal fluid (CSF) AChE as measured one day later. This corresponds to a peak of ~ 80% inhibition in CSF AChE at the time of the injection, recovering with a t1/2 of 2.4 days. Computational analyses suggest that MSF at clinically relevant doses could theoretically produce a steady-state AChE inhibition between 65% and 85% in the CNS. These data suggest that the full therapeutic advantage of AChE inhibition therapy can be realized without interference from dose-limiting gastrointestinal toxicity if an irreversible inhibitor is employed. PMID:27858711

  9. Gastrointestinal acetylcholinesterase activity following endotracheal microinstillation inhalation exposure to sarin in guinea pigs.

    PubMed

    Chanda, Soma; Song, Jian; Rezk, Peter; Sabnekar, Praveena; Doctor, Bhupendra P; Sciuto, Alfred M; Nambiar, Madhusoodana P

    2010-09-06

    The goal of this study was to assess acetylcholinesterase (AChE) inhibition at different regions of the gastrointestinal (GI) tract following inhalation exposure to nerve agent sarin. Seven major regions of the GI tract were removed from saline control animals (n=3) and 677.4 mg/m(3) sarin-exposed animals at 4h (n=4) and 24h (n=4) post-exposure. AChE activity was determined in blood and homogenized tissue supernatant by specific Ellman's assay using Iso-OMPA, a BChE inhibitor, and expressed as activity/optical density of hemoglobin for blood and activity/mg protein for tissues. Our data showed that the AChE activity was significantly decreased for groups both 4h and 24h post-sarin exposure. Among the seven chosen regions of the guinea pig GI tract, duodenum showed the highest AChE activity in control animals. The AChE activity was significantly decreased in the stomach (p=0.03), duodenum (p=0.029), jejunum (p=0.006), and ileum (p=0.006) 4h following sarin exposure. At 24h post-sarin exposure the AChE activity of duodenum (p=0.029) and ileum (p=0.006) was significantly inhibited. Esophagus showed no inhibition following sarin exposure at both 4h and 24h groups. These results suggest that the AChE activity is different in different regions of the GI tract and highest levels of AChE inhibition following sarin exposure were seen in regions exhibiting higher overall AChE activity and cholinergic function.

  10. Role of molecular isoforms of acetylcholinesterase in learning and memory functions.

    PubMed

    Das, Amitava; Dikshit, Madhu; Nath, Chandishwar

    2005-05-01

    In the present study, activity of salt soluble (SS) G1 and detergent soluble (DS) G4 molecular isoforms of acetylcholinesterase (AChE) has been investigated in rat brain areas in trained (learned), scopolamine (amnesic) and Tacrine (anti-dementic) treated rats to find out their role in learning and memory functions. AChE was estimated spectrophotometrically at 412 nm in rat brain areas. Isolation and partial purification of molecular isoforms G1 and G4 of AChE was done by gel filtration chromatography. Passive avoidance was used to test learning and memory functions. AChE activity was altered in both the fractions SS and DS of different brain areas following passive avoidance in control, scopolamine treated, tacrine treated and tacrine treatment in scopolamine pretreated rats. The peak AChE activity obtained in the DS (fraction 9) and the SS (fraction 13) fraction following gel filtration chromatography. On the basis of molecular weight fraction 9 (DS) and 13 (SS) represent the G4 and G1, respectively. The pattern of changes in the AChE activity of G1 isoform (fraction 13 of SS) and G4 isoform (fraction 9 of DS) in brain areas were similar to those of SS and DS fraction, respectively. In hippocampus, AChE activity in the fraction G1 isoform (fraction 13 of SS) was decreased only in tacrine treated rats but AChE activity in the G4 isoform (fraction 9 of DS) was decreased in both trained and tacrine treated rats. Changes in activity of G4 isoform of AChE in hippocampus could be correlated with passive avoidance learning, scopolamine induced deficit in passive avoidance and reversal of scopolamine deficit by tacrine.

  11. Neurophysiological predictors of long term response to AChE inhibitors in AD patients

    PubMed Central

    Di, L; Oliviero, A; Pilato, F; Saturno, E; Dileone, M; Marra, C; Ghirlanda, S; Ranieri, F; Gainotti, G; Tonali, P

    2005-01-01

    Background: In vivo evaluation of cholinergic circuits of the human brain has recently been introduced using a transcranial magnetic stimulation (TMS) protocol based on coupling peripheral nerve stimulation with motor cortex TMS (short latency afferent inhibition, SAI). SAI is reduced in Alzheimer's disease (AD) and drugs enhancing cholinergic transmission increase SAI. Methods: We evaluated whether SAI testing, together with SAI test-retest, after a single dose of the acetylcholinesterase (AChE) inhibitor rivastigmine, might be useful in predicting the response after 1 year treatment with rivastigmine in 16 AD patients. Results: Fourteen AD patients had pathologically reduced SAI. SAI was increased after administration of a single oral dose of rivastigmine in AD patients with abnormal baseline SAI, but individual responses to rivastigmine varied widely, with SAI change ranging from an increase in inhibition of ∼50% of test size to no change. Baseline SAI and the increase in SAI after a single dose of rivastigmine were correlated with response to long term treatment. A normal SAI in baseline conditions, or an abnormal SAI in baseline conditions that was not greatly increased by a single oral dose of rivastigmine, were invariably associated with poor response to long term treatment, while an abnormal SAI in baseline conditions in conjunction with a large increase in SAI after a single dose of rivastigmine was associated with good response to long term treatment in most of the patients. Conclusions: Evaluation of SAI may be useful for identifying AD patients likely to respond to treatment with AChE inhibitors. PMID:16024879

  12. Kinetic evidence for different mechanisms of acetylcholinesterase inhibition by (1R)- and (1S)-stereoisomers of isomalathion.

    PubMed

    Jianmongkol, S; Marable, B R; Berkman, C E; Talley, T T; Thompson, C M; Richardson, R J

    1999-02-15

    Inhibition of acetylcholinesterase (AChE) by isomalathion has been assumed to proceed by expulsion of diethyl thiosuccinyl to produce O, S-dimethyl phosphorylated AChE. If this assumption is correct, AChE inhibited by (1R)- or (1S)-isomalathions should reactivate at the same rate as AChE inhibited by configurationally equivalent (S)- or (R)-isoparathion methyl, respectively, which are expected to inhibit AChE by loss of 4-nitrophenoxyl to yield O,S-dimethyl phosphorylated AChEs. Previous work has shown that rat brain AChE inhibited by (1R)-isomalathions reactivates at the same rate as the enzyme inhibited by (S)-isoparathion methyl. However, although rat brain AChE inhibited by (R)-isoparathion methyl reactivates at a measurable rate, the enzyme inhibited by (1S)-isomalathions is intractable to reactivation. This surprising finding suggests the hypothesis that (1R)- and (1S)-stereoisomers of isomalathion inhibit AChE by different mechanisms, yielding enzymatic species distinguishable by their postinhibitory kinetics. The present study was carried out to test this hypothesis by comparing kinetic constants of reactivation (k+3) and aging (k+4) of hen brain AChE and bovine erythrocyte AChE inhibited by the four stereoisomers of isomalathion and the two stereoisomers of isoparathion methyl. Both AChEs inhibited by either (1R,3R)- or (1R,3S)-isomalathion had comparable corresponding k+3 values (spontaneous and oxime-mediated) to those of AChEs inhibited with (S)-isoparathion methyl. However, spontaneous and oxime-mediated k+3 values comparable to those of (R)-isoparathion methyl could not be obtained for AChEs inhibited by (1S,3R)- and (1S,3S)-isomalathion. Comparison of k+4 values for hen brain AChE inhibited by each stereoisomer of isomalathion and isoparathion methyl corroborated that only the (1S)-isomalathions failed to produce the expected O,S-dimethyl phosphoryl-conjugated enzymes. The results for (1R)-isomalathions suggest that the mechanism of inhibition of AChE

  13. Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 Bound at the Peripheral Site

    PubMed Central

    Bourne, Yves; Renault, Ludovic; Marchot, Pascale

    2015-01-01

    The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. PMID:25411244

  14. Revisiting the Role of Acetylcholinesterase in Alzheimer’s Disease: Cross-Talk with P-tau and β-Amyloid

    PubMed Central

    García-Ayllón, María-Salud; Small, David H.; Avila, Jesús; Sáez-Valero, Javier

    2011-01-01

    A common feature in the Alzheimer’s disease (AD) brain is the presence of acetylcholinesterase (AChE) which is commonly associated with β-amyloid plaques and neurofibrillary tangles (NFT). Although our understanding of the relationship between AChE and the pathological features of AD is incomplete, increasing evidence suggests that both β-amyloid protein (Aβ) and abnormally hyperphosphorylated tau (P-tau) can influence AChE expression. We also review recent findings which suggest the possible role of AChE in the development of a vicious cycle of Aβ and P-tau dysregulation and discuss the limited and temporary effect of therapeutic intervention with AChE inhibitors. PMID:21949503

  15. Effect of reversible ligands on oxime-induced reactivation of sarin- and cyclosarin-inhibited human acetylcholinesterase.

    PubMed

    Scheffel, Corinna; Thiermann, Horst; Worek, Franz

    2015-02-03

    Poisoning by organophosphorus compounds (OP) used as pesticides and nerve agents is due to irreversible inhibition of the enzyme acetylcholinesterase (AChE). Oximes have been widely recognized for their potency to reactivate the inhibited enzyme. The limited efficacy of currently available oximes against a broad spectrum of OP-compounds initiated novel research efforts to improve oxime-based treatment. Hereby, oxime-induced reactivation of OP-inhibited non-human AChE was reported to be accelerated by different AChE-ligands. To investigate this concept with AChE from human source, the inhibitory potency, binding properties and the potential enhancement of oxime-induced reactivation of OP-inhibited AChE by structurally different AChE-ligands was assessed. Several ligands competed with the oxime for the AChE binding-site impairing reactivation of OP-inhibited AChE whereas a markedly accelerated reactivation of sarin-inhibited enzyme by obidoxime was recorded in the presence of edrophonium, galanthamine and donepezil. Enhancement of oxime-induced reactivation with ligands was presumably subject to prevention of re-inhibition by the reaction product phosphonyloxime (POX). In the end, the results of the present study did not confirm that AChE-ligands directly accelerate the reactivation of OP-inhibited AChE by oximes, but indirectly by prevention of re-inhibition by the reaction product POX. This may be due to different experimental conditions and species differences between human and non-human AChE of previous experiments with non-human AChE.

  16. Inhibition of acetylcholinesterase by Tea Tree oil.

    PubMed

    Mills, Clive; Cleary, Brian J; Gilmer, John F; Walsh, John J

    2004-03-01

    Pediculosis is a widespread condition reported in schoolchildren. Treatment most commonly involves the physical removal of nits using fine-toothcombs and the chemical treatment of adult lice and eggs with topical preparations. The active constituents of these preparations frequently exert their effects through inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Increasing resistance to many preparations has led to the search for more effective treatments. Tea Tree oil, otherwise known as Melaleuca oil, has been added to several preparations as an alternative treatment of head lice infestations. In this study two major constituents of Tea Tree oil, 1,8-cineole and terpinen-4-ol, were shown to inhibit acetylcholinesterase at IC50 values (inhibitor concentrations required to give 50% inhibition) of 0.04 and 10.30 mM, respectively. Four samples of Tea Tree oil tested (Tisserand, Body Treats, Main Camp and Irish Health Culture Association Pure Undiluted) showed anticholinesterase activity at IC50 values of 0.05, 0.10, 0.08 and 0.11 microL mL(-1), respectively. The results supported the hypothesis that the insecticidal activity of Tea Tree oil was attributable, in part, to the anticholinesterase activity of Tea Tree oil.

  17. Acetylcholinesterase in Biofouling Species: Characterization and Mode of Action of Cyanobacteria-Derived Antifouling Agents

    PubMed Central

    Almeida, Joana R.; Freitas, Micaela; Cruz, Susana; Leão, Pedro N.; Vasconcelos, Vitor; Cunha, Isabel

    2015-01-01

    Effective and ecofriendly antifouling (AF) compounds have been arising from naturally produced chemicals. The objective of this study is to use cyanobacteria-derived agents to investigate the role of acetylcholinesterase (AChE) activity as an effect and/or mode of action of promising AF compounds, since AChE inhibitors were found to inhibit invertebrate larval settlement. To pursue this objective, in vitro quantification of AChE activity under the effect of several cyanobacterial strain extracts as potential AF agents was performed along with in vivo AF (anti-settlement) screening tests. Pre-characterization of different cholinesterases (ChEs) forms present in selected tissues of important biofouling species was performed to confirm the predominance of AChE, and an in vitro AF test using pure AChE activity was developed. Eighteen cyanobacteria strains were tested as source of potential AF and AChE inhibitor agents. Results showed effectiveness in selecting promising eco-friendly AF agents, allowing the understanding of the AF biochemical mode of action induced by different compounds. This study also highlights the potential of cyanobacteria as source of AF agents towards invertebrate macrofouling species. PMID:26213967

  18. In vitro acetylcholinesterase inhibition by psoralen using molecular docking and enzymatic studies

    PubMed Central

    Somani, Gauresh; Kulkarni, Chinmay; Shinde, Prashant; Shelke, Rupesh; Laddha, Kirti; Sathaye, Sadhana

    2015-01-01

    Introduction: Alzheimer's disease (AD) has increased at an alarming rate and is now a worldwide health problem. Inhibitors of acetylcholinesterase (AChE) leading to inhibition of acetylcholine breakdown constitute the main therapeutic strategy for AD. Psoralen was investigated as inhibitor of AChE enzyme in an attempt to explore its potential for the management of AD. Materials and Methods: Psoralen was isolated from powdered Psoralea corylifolia fruits. AChE enzyme inhibitory activity of different concentrations of psoralen was investigated by use of in vitro enzymatic and molecular docking studies. Further, the enzyme kinetics were studied using Lineweaver-Burk plot. Results: Psoralen was found to inhibit AChE enzyme activity in a concentration-dependent manner. Kinetic studies showed psoralen inhibits AChE in a competitive manner. Molecular docking study revealed that psoralen binds well within the binding site of the enzyme showing interactions such as π-π stacking and hydrogen bonding with residues present therein. Conclusion: The result of AChE enzyme inhibitory activity of the psoralen in this study is promising. It could be further explored as a potential candidate for further development of new drugs against AD. PMID:25709334

  19. Three acetylcholinesterases of the pinewood nematode, Bursaphelenchus xylophilus: insights into distinct physiological functions.

    PubMed

    Kang, Jae Soon; Lee, Dae-Weon; Choi, Jae Young; Je, Yeon Ho; Koh, Young Ho; Lee, Si Hyeock

    2011-02-01

    Acetylcholinesterase (AChE) plays a key role in postsynaptic transmission in most animals. Nematodes encode multiple AChEs, implying its functional diversity. To explore physiological functions of multiple AChEs, three distinct AChEs (BxACE-1, BxACE-2, and BxACE-3) were identified and characterized from the pinewood nematode. Sequencing comparison with Torpedo AChE and Caenorhabditis elegans ACEs identified choline-binding site, catalytic triad functional site, three internal disulfide bonds and aromatic residues for the catalytic gorge. Transcriptional profiling by quantitative real-time PCR revealed that BxACE-3 is more actively transcribed than BxACE-1 (2-3 times) and BxACE-2 (9-18 times) in both propagative and dispersal stages. The three BxACEs were functionally expressed using baculovirus system. Kinetic analysis of in vitro-expressed BxACEs revealed that the substrate specificity was highest in BxACE-1 whereas the catalytic efficiency was highest in BxACE-2. In inhibition assay, BxACE-3 showed the lowest inhibition rate. Taken together, it appears that both BxACE-1 and BxACE-2 play common but non-overlapping roles in synaptic transmission, whereas BxACE-3 may have non-neuronal functions. The current findings should provide valuable insights into the evolutionary process and various physiological roles of AChE.

  20. Nanomaterials - Acetylcholinesterase Enzyme Matrices for Organophosphorus Pesticides Electrochemical Sensors: A Review

    PubMed Central

    Periasamy, Arun Prakash; Umasankar, Yogeswaran; Chen, Shen-Ming

    2009-01-01

    Acetylcholinesterase (AChE) is an important cholinesterase enzyme present in the synaptic clefts of living organisms. It maintains the levels of the neurotransmitter acetylcholine by catalyzing the hydrolysis reaction of acetylcholine to thiocholine. This catalytic activity of AChE is drastically inhibited by trace amounts of organophosphorus (OP) pesticides present in the environment. As a result, effective monitoring of OP pesticides in the environment is very desirable and has been done successfully in recent years with the use of nanomaterial-based AChE sensors. In such sensors, the enzyme AChE has been immobilized onto nanomaterials like multiwalled carbon nanotubes, gold nanoparticles, zirconia nanoparticles, cadmium sulphide nano particles or quantum dots. These nanomaterial matrices promote significant enhancements of OP pesticide determinations, with the thiocholine oxidation occurring at much lower oxidation potentials. Moreover, nanomaterial-based AChE sensors with rapid response, increased operational and long storage stability are extremely well suited for OP pesticide determination over a wide concentration range. In this review, the unique advantages of using nanomaterials as AChE immobilization matrices are discussed. Further, detection limits, sensitivities and correlation coefficients obtained using various electroanalytical techniques have also been compared with chromatographic techniques. PMID:22408512

  1. Carbon-11 labeling of CP-126,998*: A radiotracer for in vivo studies of acetylcholinesterase

    SciTech Connect

    Musachio, J.L.; Flesher, J.E.; Scheffel, U.

    1996-05-01

    The study of acetylcholinesterase (AChE) via PET is of interest as reduced activity of this enzyme has been observed in Alzheimer`s disease. Our efforts to develop a radiotracer for mapping of AChE have focused on the N-benzylpiperidine benzisoxazole, CP-126,998, a highly potent (IC{sub 50}=0.48 nm) and selective inhibitor of AChE. High specific activity [C-11] CP-126,998 was synthesized (14 - 24% radiochemical yield, non-decay corrected) by treatment of the desmethyl precursor, CP-118,954, with [C-11] methyl iodide and tetrabutylammonium hydroxide in DMF. In vivo studies with [C-11] CP-126,998 in mice show that this radiotracer displays highest uptake in striatum (6.2 %ID/g), a brain region known to be rich in AChE. The (striatum-cerebellum)/cerebellar radioactivity ratio reached a maximum of 4.3 at 30 min postinjection, and this ratio decreased to 2.4 at 120 min. .Radiotracer binding was saturable in vivo by pretreatment with CP-118,954. Pretreatment of mice with diisopropylfluorophosphate (4 mg/kg i.p.), a known AChE inhibitor, significantly inhibited binding in striatum in a dose-dependent manner. Initial results suggest that [C-11] CP-126,998 may prove useful as a marker for the study of AChE in humans via PET.

  2. Galangin, a flavonol derived from Rhizoma Alpiniae Officinarum, inhibits acetylcholinesterase activity in vitro.

    PubMed

    Guo, Ava J Y; Xie, Heidi Q; Choi, Roy C Y; Zheng, Ken Y Z; Bi, Cathy W C; Xu, Sherry L; Dong, Tina T X; Tsim, Karl W K

    2010-09-06

    Acetylcholinesterase (AChE) inhibitors are widely used for the treatment of Alzheimer's disease (AD). Several AChE inhibitors, e.g. rivastigmine, galantamine and huperzine are originating from plants, suggesting that herbs could potentially serve as sources for novel AChE inhibitors. Here, we searched potential AChE inhibitors from flavonoids, a group of naturally occurring compounds in plants or traditional Chinese medicines (TCM). Twenty-one flavonoids, covered different subclasses, were tested for their potential function in inhibiting AChE activity from the brain in vitro. Among all the tested flavonoids, galangin, a flavonol isolated from Rhizoma Alpiniae Officinarum, the rhizomes of Alpiniae officinarum (Hance.) showed an inhibitory effect on AChE activity with the highest inhibition by over 55% and an IC(50) of 120 microM and an enzyme-flavonoid inhibition constant (K(i)) of 74 microM. The results suggest that flavonoids could be potential candidates for further development of new drugs against AD.

  3. Acetylcholinesterase in Biofouling Species: Characterization and Mode of Action of Cyanobacteria-Derived Antifouling Agents.

    PubMed

    Almeida, Joana R; Freitas, Micaela; Cruz, Susana; Leão, Pedro N; Vasconcelos, Vitor; Cunha, Isabel

    2015-07-24

    Effective and ecofriendly antifouling (AF) compounds have been arising from naturally produced chemicals. The objective of this study is to use cyanobacteria-derived agents to investigate the role of acetylcholinesterase (AChE) activity as an effect and/or mode of action of promising AF compounds, since AChE inhibitors were found to inhibit invertebrate larval settlement. To pursue this objective, in vitro quantification of AChE activity under the effect of several cyanobacterial strain extracts as potential AF agents was performed along with in vivo AF (anti-settlement) screening tests. Pre-characterization of different cholinesterases (ChEs) forms present in selected tissues of important biofouling species was performed to confirm the predominance of AChE, and an in vitro AF test using pure AChE activity was developed. Eighteen cyanobacteria strains were tested as source of potential AF and AChE inhibitor agents. Results showed effectiveness in selecting promising eco-friendly AF agents, allowing the understanding of the AF biochemical mode of action induced by different compounds. This study also highlights the potential of cyanobacteria as source of AF agents towards invertebrate macrofouling species.

  4. The exploration of thienothiazines as selective butyrylcholinesterase inhibitors.

    PubMed

    Karlsson, Daniela; Fallarero, Adyary; Brunhofer, Gerda; Mayer, Claudia; Prakash, Ohm; Mohan, C Gopi; Vuorela, Pia; Erker, Thomas

    2012-08-30

    The role of butyrylcholinesterase (BChE) in the progression of Alzheimer's disease (AD) has recently become more crucial. In the AD brain, selective BChE inhibitors have been demonstrated to have a beneficial effect in vivo, probably by recovering cholinergic activity and/or by restoring AChE:BChE activity ratios to the levels observed in the healthy brain. Thienothiazines are compounds sharing some structural features with phenothiazines, which are known to be potent BChE inhibitors. Thus, in this contribution 45 thienothiazines were investigated for their BChE inhibitory activity. Six of them were proven to be potent and selective inhibitors of equine BChE's hydrolase activity. Structure-activity relationships were laid out, and a tentative pharmacophore model for BChE inhibitors of the thienothiazine type was proposed. The most active compound, 3f, displayed a mixed type of inhibition and was also active against the human BChE (huBChE) with an IC(50) huBChE of 0.51 ± 0.07 μM. Computational studies suggested that 3f likely binds to the catalytic site and nearby to the peripheral site of the huBChE in an extended form. In addition, the chemical space occupied by the active thienothiazines, as opposed to phenothiazines and other representative chemical classes of BChE inhibitors, was explored with the aid of ChemGPS-NP, and the relevant chemical space regions were identified. This study shows for the first time that thienothiazines represent a new group of BChE inhibitors that can be used as molecular probes for studying the role of BChE in the brain or for developing newer drug leads for AD therapy.

  5. The protective role of tacrine and donepezil in the retina of acetylcholinesterase knockout mice

    PubMed Central

    Yi, Yun-Min; Cai, Li; Shao, Yi; Xu, Man; Yi, Jing-Lin

    2015-01-01

    AIM To determine the effect of different concentrations of the acetylcholinesterase (AChE) inhibitors tacrine and donepezil on retinal protection in AChE+/− mice (AChE knockout mice) of various ages. METHODS Cultured ARPE-19 cells were treated with hydrogen peroxide (H2O2) at concentrations of 0, 250, 500, 1000 and 2000 µmol/L and protein levels were measured using Western blot. Intraperitoneal injections of tacrine and donepezil (0.1 mg/mL, 0.2 mg/mL and 0.4 mg/mL) were respectively given to AChE+/− mice aged 2mo and 4mo and wild-type S129 mice for 7d; phosphate buffered saline (PBS) was administered to the control group. The mice were sacrificed after 30d by in vitro cardiac perfusion and retinal samples were taken. AChE-deficient mice were identified by polymerase chain reaction (PCR) analysis using specific genotyping protocols obtained from the Jackson Laboratory website. H&E staining, immunofluorescence and Western blot were performed to observe AChE protein expression changes in the retinal pigment epithelial (RPE) cell layer. RESULTS Different concentrations of H2O2 induced AChE expression during RPE cell apoptosis. AChE+/− mice retina were thinner than those in wild-type mice (P<0.05); the retinal structure was still intact at 2mo but became thinner with increasing age (P<0.05); furthermore, AChE+/− mice developed more slowly than wild-type mice (P<0.05). Increased concentrations of tacrine and donepezil did not significantly improve the protection of the retina function and morphology (P>0.05). CONCLUSION In vivo, tacrine and donepezil can inhibit the expression of AChE; the decrease of AChE expression in the retina is beneficial for the development of the retina. PMID:26558196

  6. Neurotoxicology of bis(n)-tacrines on Blattella germanica and Drosophila melanogaster acetylcholinesterase.

    PubMed

    Mutunga, James M; Boina, Dhana Raj; Anderson, Troy D; Bloomquist, Jeffrey R; Carlier, Paul R; Wong, Dawn M; Lam, Polo C-H; Totrov, Maxim M

    2013-08-01

    A series of bis(n)-tacrines were used as pharmacological probes of the acetylcholinesterase (AChE) catalytic and peripheral sites of Blattella germanica and Drosophila melanogaster, which express AChE-1 and AChE-2 isoforms, respectively. In general, the potency of bis(n)-tacrines was greater in D. melanogaster AChE (DmAChE) than in B. germanica AChE (BgAChE). The change in potency with tether length was high in DmAChE and low in BgAChE, associated with 90-fold and 5.2-fold maximal potency gain, respectively, compared to the tacrine monomer. The optimal tether length for Blattella was 8 carbons and for Drosophila was 10 carbons. The two species differed by only about twofold in their sensitivity to tacrine monomer, indicating that differential potency occurred among dimeric bis(n)-tacrines due to structural differences in the peripheral site. Multiple sequence alignment and in silico homology modeling suggest that aromatic residues of DmAChE confer higher affinity binding, and the lack of same at the BgAChE peripheral site may account, at least in part, to the greater overall sensitivity of DmAChE to bis(n)-tacrines, as reflected by in vitro assay data. Topical and injection assays in cockroaches found minimal toxicity of bis(n)-tacrines. Electrophysiological studies on D. melanogaster central nervous system showed that dimeric tacrines do not readily cross the blood brain barrier, explaining the observed nonlethality to insects. Although the bis(n)-tacrines were not good insecticide candidates, the information obtained in this study should aid in the design of selective bivalent ligands targeting insect, pests, and disease vectors.

  7. Effect of thermal stress and water deprivation on the acetylcholinesterase activity of the pig brain and hypophyses

    NASA Astrophysics Data System (ADS)

    Adejumo, D. O.; Egbunike, G. N.

    1988-06-01

    The effects of direct exposure of boars to thermal stress for 1 h daily for 5 days and to acute water deprivation for 24 or 48 h were studied on the acetylcholinesterase (AChE) activity of porcine brain and hypophysial regions. Mean ambient temperatures, respiratory rates and rectal temperatures in the open were significantly higher than inside the pen. Heat stress induced a rise in AChE activities in the pons, cerebellum, amygdala, hippocampus, hypothalamus, mid-brain and medulla oblongata. However, no significant changes were observed in the cerebral cortex, adenohypophysis and neurohypophysis. Water deprivation significantly ( P<0.05) depressed AChE activity to varying extents depending on the duration of water restriction. Thus AChE activity in the amygdala was depressed by water deprivation for 24 h but partially restored at 48 h. The pons and medulla oblongata were comparable to the amygdala in this respect. The adenohypophysis and neurohypophysis were relatively unaffected.

  8. Use and disuse and the control of acetylcholinesterase activity in fast and slow twitch muscle of rat

    NASA Technical Reports Server (NTRS)

    Dettbarn, W. D.; Groswald, D.; Gupta, R. C.; Misulis, K. E.

    1985-01-01

    The role of acetylcholinesterase (AChE) in neuromuscular transmission is relatively well established, little is known, however, of the mechanisms that regulate its synthesis and control its specific distribution in fast and slow muscle. Innervation plays an important role in the regulation of AChE and elimination of the influence of the nerve by surgical denervation results in a loss of AChE. The influences of the nerve and how they are mediated was investigated. It is suggested that muscle usage and other factors such as materials carried by axonal transport may participate in the regulation of this enzyme. The mechanisms that regulate AChE and its molecular forms in two functionally different forms are studied.

  9. Novel bis-(−)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property

    SciTech Connect

    Zheng, Wei; Li, Juan; Qiu, Zhuibai; Xia, Zheng; Li, Wei; Yu, Lining; Chen, Hailin; Chen, Jianxing; Chen, Yan; Hu, Zhuqin; Zhou, Wei; Shao, Biyun; Cui, Yongyao; Xie, Qiong; Chen, Hongzhuan

    2012-10-01

    The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(−)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC{sub 50} values of 9.63 μM (for ZLA) and 8.64 μM (for ZLB), and prevent AChE-induced amyloid-β (Aβ) aggregation with IC{sub 50} values of 49.1 μM (for ZLA) and 55.3 μM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aβ aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD. -- Highlights: ► Two novel bis-(−)-nor-meptazinol derivatives are designed and synthesized. ► ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency. ► They are potential leads for disease-modifying treatment of Alzheimer's disease.

  10. Lower Acetylcholinesterase Activity among Children Living with Flower Plantation Workers

    PubMed Central

    Suarez-Lopez, Jose R.; Jacobs, David R.; Himes, John H.; Alexander, Bruce H.; Lazovich, DeAnn; Gunnar, Megan

    2012-01-01

    BACKGROUND Children of workers exposed to pesticides are at risk of secondary pesticide exposure. We evaluated the potential for lower acetylcholinesterase activity in children cohabiting with fresh-cut flower plantation workers, which would be expected from organophosphate and carbamate insecticide exposure. Parental home surveys were performed and acetylcholinesterase activity was measured in 277 children aged 4–9 years in the study of Secondary Exposure to Pesticides among Infants, Children and Adolescents (ESPINA). Participants lived in a rural county in Ecuador with substantial flower plantation activity. RESULTS Mean acetylcholinesterase activity was 3.14 U/ml, standard deviation (SD): 0.49. It was lower by 0.09 U/ml (95% confidence interval (CI) −0.19, −0.001) in children of flower workers (57% of participants) than non-flower workers’ children, after adjustment for gender, age, height-for-age, hemoglobin concentration, income, pesticide use within household lot, pesticide use by contiguous neighbors, examination date and residence distance to nearest flower plantation. Using a 4 level polychotomous acetylcholinesterase activity dependent variable, flower worker cohabitation (vs. not) had odds ratio 3.39 (95% CI 1.19, 9.64) for being <15th percentile compared to the highest tertile. Children cohabitating for ≥5 years (vs. never) had OR of 4.11 (95% CI: 1.17, 14.38) of AChE activity within <15th percentile compared to the highest tertile. CONCLUSIONS Cohabitation with a flower worker was related to lower acetylcholinesterase activity in children. This supports the hypothesis that the amount of take-home pesticides from flower workers suffices to decrease acetylcholinesterase activity, with lower activity associated with longer exposure. PMID:22405996

  11. Virtual screening discovery of new acetylcholinesterase inhibitors issued from CERMN chemical library.

    PubMed

    Sopkova-de Oliveira Santos, Jana; Lesnard, Aurelien; Agondanou, Jean-Hugues; Dupont, Nathalie; Godard, Anne-Marie; Stiebing, Silvia; Rochais, Christophe; Fabis, Frederic; Dallemagne, Patrick; Bureau, Ronan; Rault, Sylvain

    2010-03-22

    In our quest to find new inhibitors able to inhibit acetylcholinesterase (AChE) and, at the same time, to protect neurons from beta amyloid toxicity, i.e., inhibitors interacting with the catalytic anionic subsite as well as with the peripherical anionic site of AChE, a virtual screening of the Centre d'Etudes et de Recherche sur le Medicament de Normandie (CERMN) chemical library was carried out. Two complementary approaches were applied, i.e., a ligand- and a structure-based screening. Each screening led to the selection of different compounds, but only two were present in both screening results. In vitro tests on AChE showed that one of those compounds presented a very good inhibition activity, of the same order as Donepezil. This result shows the real complementary of both methods for the discovery of new ligands.

  12. Ruthenium(II) polypyridyl complex as inhibitor of acetylcholinesterase and Aβ aggregation.

    PubMed

    Vyas, Nilima A; Bhat, Satish S; Kumbhar, Avinash S; Sonawane, Uddhavesh B; Jani, Vinod; Joshi, Rajendra R; Ramteke, Shefali N; Kulkarni, Prasad P; Joshi, Bimba

    2014-03-21

    Two ruthenium(II) polypyridyl complexes [Ru(phen)3](2+) (1) and [Ru(phen)2(bxbg)](2+) (2) (where phen = 1,10 phenanthroline, bxbg = bis(o-xylene)bipyridine glycoluril) have been evaluated for acetylcholinesterase (AChE) and Amyloid-β peptide (Aβ) aggregation inhibition. Complex 2 exhibits higher potency of AChE inhibition and kinetics and molecular modeling studies indicate that ancillary ligand plays significant role in inhibitory potency exhibited by complex 2. The inhibitory effect of these complexes on Aβ (1-40) aggregation is investigated using Thioflavin T fluorescence and Transmission Electron Microscopy. Both complexes efficiently inhibit Aβ (1-40) aggregation and are negligibly toxic to human neuroblastoma cells. This is the first demonstration that ruthenium(II) polypyridyl complexes simultaneously inhibit AChE and Aβ aggregation.

  13. Applications of Integrated Data Mining Methods to Exploring Natural Product Space for Acetylcholinesterase Inhibitors

    PubMed Central

    Schuster, Daniela; Kern, Lisa; Hristozov, Dimitar P.; Terfloth, Lothar; Bienfait, Bruno; Laggner, Christian; Kirchmair, Johannes; Grienke, Ulrike; Wolber, Gerhard; Langer, Thierry; Stuppner, Hermann; Gasteiger, Johann; Rollinger, Judith M.

    2013-01-01

    Nature, especially the plant kingdom, is a rich source for novel bioactive compounds that can be used as lead compounds for drug development. In order to exploit this resource, the two neural network-based virtual screening techniques novelty detection with self-organizing maps (SOMs) and counterpropagation neural network were evaluated as tools for efficient lead structure discovery. As application scenario, significant descriptors for acetylcholinesterase (AChE) inhibitors were determined and used for model building, theoretical model validation, and virtual screening. Top-ranked virtual hits from both approaches were docked into the AChE binding site to approve the initial hits. Finally, in vitro testing of selected compounds led to the identification of forsythoside A and (+)-sesamolin as novel AChE inhibitors. PMID:20214575

  14. Nantenine as an acetylcholinesterase inhibitor: SAR, enzyme kinetics and molecular modeling investigations

    PubMed Central

    Pecic, Stevan; McAnuff, Marie A.; Harding, Wayne W.

    2015-01-01

    Nantenine, as well as a number of flexible analogs, were evaluated for acetylcholinesterase (AChE) inhibitory activity in microplate spectrophotometric assays based on Ellman’s method. It was found that the rigid aporphine core of nantenine is an important structural requirement for its anticholinesterase activity. Nantenine showed mixed inhibition kinetics in enzyme assays. Molecular docking experiments suggest that nantenine binds preferentially to the catalytic site of AChE but is also capable of interacting with the peripheral anionic site (PAS) of the enzyme, thus accounting for its mixed inhibition profile. The aporphine core of nantenine may thus be a useful template for the design of novel PAS or dual-site AChE inhibitors. Inhibiting the PAS is desirable for prevention of aggregation of the amyloid peptide Aβ, a major causative factor in the progression of Alzheimer’s disease (AD). PMID:20583856

  15. Highly Sensitive and Selective Immuno-capture/Electrochemical Assay of Acetylcholinesterase Activity in Red Blood Cells: A Biomarker of Exposure to Organophosphorus Pesticides and Nerve Agents

    SciTech Connect

    Chen, Aiqiong; Du, Dan; Lin, Yuehe

    2012-02-09

    Acetylcholinesterase (AChE) enzyme activity in red blood cells (RBCs) is a useful biomarker for biomonitoring of exposures to organophosphorus (OP) pesticides and chemical nerve agents. In this paper, we reported a new method for AChE activity assay based on selective immuno-capture of AChE from biological samples followed by enzyme activity assay of captured AChE using a disposable electrochemical sensor. The electrochemical sensor is based on multiwalled carbon nanotubes-gold nanocomposites (MWCNTs-Au) modified screen printed carbon electrode (SPCE). Upon the completion of immunoreaction, the target AChE (including active and inhibited) is captured onto the electrode surface and followed by an electrochemical detection of enzymatic activity in the presence of acetylthiocholine. A linear response is obtained over standard AChE concentration range from 0.1 to 10 nM. To demonstrate the capability of this new biomonitoring method, AChE solutions dosed with different concentration of paraoxon were used to validate the new AChE assay method. AChE inhibition in OP dosed solutions was proportional to its concentration from 0.2 to 50 nM. The new AChE activity assay method for biomonitoring of OP exposure was further validated with in-vitro paraoxon-dosed RBC samples. The established electrochemical sensing platform for AChE activity assay not only avoids the problem of overlapping substrate specificity with esterases by using selective antibody, but also eliminates potential interference from other electroactive species in biological samples. It offers a new approach for sensitive, selective, and rapid AChE activity assay for biomonitoring of exposures to OPs.

  16. Effects of Anabaena spiroides (Cyanobacteria) aqueous extracts on the acetylcholinesterase activity of aquatic species.

    PubMed

    Monserrat, J M; Yunes, J S; Bianchini, A

    2001-06-01

    The effects of aqueous extracts from a cyanobacteria species, Anabaena spiroides, on fish (Odontesthes argentinensis), crab (Callinectes sapidus), and purified eel acetylcholinesterase (AChE) activity were studied. In vitro concentrations of A. spiroides aqueous extract that inhibited 50% of enzyme activity (IC50) were 23.0, 17.2, and 45.0 mg/L of lyophilized cyanobacteria for eel, fish, and crab AChE, respectively. Eel AChE inhibition follows pseudo-first-order kinetics, the same expected for organophosphorus pesticides. Inhibition of purified eel AChE using mixtures of bioxidized malathion and aqueous extract of A. spiroides showed a competitive feature (p < 0.05), suggesting that the toxin(s) could be structurally similar to an organophosphorus pesticide and that toxins present in the aqueous extract inhibit the active site of the enzyme. The inhibition recovery assays using 2-PAM (0.3 mM) showed that (1) bioxidized malathion inhibited 27.0 +/- 1.1% of crab and 36.5 +/- 0.1% of eel AChE activities; (2) with bioxidized malathion + 2-PAM the registered inhibition was 13.2 +/- 2.1% and 3.7 +/- 0.5% in crab and eel AChE, respectively; (3) the aqueous extract from A. spiroides inhibited 17.4 +/- 2.2% and 59.9 +/- 0.5% of crab and eel AChE activity, respectively; and (4) aqueous extract + 2-PAM inhibited 22.3 +/- 2.6 and 61.5 +/- 0.2% of crab and eel AChEs. The absence of enzyme activity recovery after 2-PAM exposure could imply that the enzyme aging process was extremely quick.

  17. Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: Effect of glycyl-l-glutamine. (Reannouncement with new availability information)

    SciTech Connect

    Yourick, J.J.; Eklo, P.A.; McCluskey, M.P.; Ray, R.

    1991-12-31

    Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 x 10-6 M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10-6, 10-5, or 10.4 M) or glycyl-L-glutamic acid (10-6 M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChe activity gradually increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman.

  18. AChE Inhibition-based Multi-target-directed Ligands, a Novel Pharmacological Approach for the Symptomatic and Disease-modifying Therapy of Alzheimer's Disease

    PubMed Central

    Wang, Yu; Wang, Hao; Chen, Hong-zhuan

    2016-01-01

    Alzheimer's disease (AD) is the most common form of dementia in elder people, characterised by a progressive decline in memory as a result of an impairment of cholinergic neurotransmission. To date acetylcholinesterase inhibitors (AChEIs) have become the most prescribed drugs for the symptomatic treatment of mild to moderate AD. However, the traditional “one molecule-one target” paradigm is not sufficient and appropriate to yield the desired therapeutic efficacy since multiple factors, such as amyloid-β (Aβ) deposits, neuroinflammation, oxidative stress, and decreased levels of acetylcholine (ACh) have been thought to play significant roles in the AD pathogenesis. New generation of multi-target drugs is earnestly demanded not only for ameliorating symptoms but also for modifying the disease. Herein, we delineated the catalytic and non-catalytic functions of AChE, and summarized the works of our group and others in research and development of novel AChEI-based multi-target-directed ligands (MTDLs), such as dual binding site AChEIs and multi-target AChEIs inhibiting Aβ aggregation, regulating Aβ procession, antagonizing platelet-activating factor (PAF) receptor, scavenging oxygen radical, chelating metal ions, inhibiting monoamine oxidase B (MAO-B), blocking N-methyl-D-aspartic acid (NMDA) receptor and others. PMID:26786145

  19. Inhibitory Effects of Sodium Arsenite and Acacia Honey on Acetylcholinesterase in Rats

    PubMed Central

    Odunola, Oyeronke A.; Gbadegesin, Michael A.; Sallau, Abdullahi B.; Ndidi, Uche S.; Ibrahim, Mohammed A.

    2015-01-01

    This study was conducted to investigate the effect of sodium arsenite and Acacia honey on acetylcholinesterase (AChE) activity and electrolytes in the brain and serum of Wistar rats. Male Wistar albino rats in four groups of five rats each were treated with distilled water, sodium arsenite (5 mg/kg body weight), Acacia honey (20% v/v), and sodium arsenite and Acacia honey, daily for one week. The sodium arsenite and Acacia honey significantly (P < 0.05) decreased AChE activity in the brain with the combined treatment being more potent. Furthermore, sodium arsenite and Acacia honey significantly (P < 0.05) decreased AChE activity in the serum. Strong correlation was observed between the sodium and calcium ion levels with acetylcholinesterase activity in the brain and serum. The gas chromatography mass spectrometry analysis of Acacia honey revealed the presence of a number of bioactive compounds such as phenolics, sugar derivatives, and fatty acids. These findings suggest that sodium arsenite and/or Acacia honey modulates acetylcholinesterase activities which may be explored in the management of Alzheimer's diseases but this might be counteracted by the hepatotoxicity induced by arsenics. PMID:25821630

  20. Is acetylcholinesterase a biomarker of susceptibility in Daphnia magna (Crustacea, Cladocera) after deltamethrin exposure?

    PubMed

    Toumi, Héla; Boumaiza, Moncef; Millet, Maurice; Radetski, Claudemir Marcos; Felten, Vincent; Férard, Jean François

    2015-02-01

    In the present study, we explored the possibility of using the acetylcholinesterase (AChE) as a biomarker after deltamethrin (pyrethroid insecticide) exposure with three strains of the cladoceran Daphnia magna. Four calculated time-weighted deltamethrin concentrations (20.1, 40.3, 80.6 and 161.3 ng L(-1)) were compared against control acetylcholinesterase activity. Our results showed that after 48 h of deltamethrin exposure, all treatments induced a significant decrease of AChE activities whatever the three considered strains. However, diverse responses were registered in terms of lowest observed effect concentrations (LOEC: 80.6 ng L(-1) for strain 1 and 20.1 ng L(-1) for strains 2 and 3) revealing differences in sensitivity among the three tested strains of D. magna. Our results suggest that after deltamethrin exposure, the AChE activity responses can be also used as a biomarker of susceptibility (i.e., variation of strain specific response). Moreover, our results show that strain 1 is the less sensitive in terms of IC50-48 h of AChE, whereas it became the most sensitive when considering the EC50-48 h estimated in the standard ecotoxicity test.

  1. [Achetylcholinesterase (AChE) inhibition and serum lipokines in Alzheimer's disease: friend or foe?].

    PubMed

    Kovacs, Janos; Pakaski, Magdolna; Juhasz, Anna; Feher, Agnes; Drotos, Gergely; Fazekas, Csilla Orsike; Horvath, Tamas Laszlo; Janka, Zoltan; Kalman, Janos

    2012-03-01

    Throughout the natural progression of Alzheimer's disease (AD), the body mass index (BMI) decreases. This is believed to be brought on by the disturbance in the central lipid metabolism, but the exact mechanism is yet unknown. Adipokines (adiponectin, leptin), hormones produced by the adipose tissue, change glucose and lipid metabolism, and have an anorectic effect through increasing energy consumption in the hypothalamus. The goal of our study was to examine donepezil - an acetylcholinesterase inhibitor (AChEI) currently used in AD therapy -, and to what degree it influences the serum adipokine levels and metabolic parameters of AD patients. During the self-evaluation of 26 clinically diagnosed mild to moderate AD patients, therapy with 10 mg/day donepezil was started according to current protocols. We measured serum adiponectin, leptin, LDL, HDL, trigliceride levels, and BMI and ApoE polymorphism at the beginning of our study, and at 3 and 6-months intervals respectively. All data were analyzed with SPSS 17. In comparison with pre-donepezil therapy values, at the third month interval serum adiponectin levels showed an increasing and leptin levels a decreasing tendency. At the six month interval, adiponectin levels significantly increased (p=0.007), leptin levels decreased (p=0.013), BMI (p=0.001) and abdominal circumference (p=0.017) was significantly lower at 6 months as compared to control values. We did not observe any changes in the lipid profile, and ApoE4 allele carrying showed no association with the parameters. To our knowledge, we are the first to publish that AChEI therapy with donepezil alters lipokine levels, which positively influences the currently known pathomechanism and numerous risk factors of AD. The AChEI treatment-induced weight loss should be considered in the long-term therapy of AD patients.

  2. The reactivation of tabun-inhibited mutant AChE with Ortho-7: steered molecular dynamics and quantum chemical studies.

    PubMed

    Lo, Rabindranath; Chandar, Nellore Bhanu; Ghosh, Shibaji; Ganguly, Bishwajit

    2016-04-01

    A highly toxic nerve agent, tabun, can inhibit acetylcholinesterase (AChE) at cholinergic sites, which leads to serious cardiovascular complications, respiratory compromise and death. We have examined the structural features of the tabun-conjugated AChE complex with an oxime reactivator, Ortho-7, to provide a strategy for designing new and efficient reactivators. Mutation of mAChE within the choline binding site by Y337A and F338A and its interaction with Ortho-7 has been investigated using steered molecular dynamics (SMD) and quantum chemical methods. The overall study shows that after mutagenesis (Y337A), the reactivator can approach more freely towards the phosphorylated active site of serine without any significant steric hindrance in the presence of tabun compared to the wild type and double mutant. Furthermore, the poor binding of Ortho-7 with the peripheral residues of mAChE in the case of the single mutant compared to that of the wild-type and double mutant (Y337A/F338A) can contribute to better efficacy in the former case. Ortho-7 has formed a greater number of hydrogen bonds with the active site surrounding residues His447 and Phe295 in the case of the single mutant (Y337A), and that stabilizes the drug molecule for an effective reactivation process. The DFT M05-2X/6-31+G(d) level of theory shows that the binding energy of Ortho-7 with the single mutant (Y337A) is energetically more preferred (-19.8 kcal mol(-1)) than the wild-type (-8.1 kcal mol(-1)) and double mutant (Y337A/F338A) (-16.0 kcal mol(-1)). The study reveals that both the orientation of the oxime reactivator for nucleophilic attack and the stabilization of the reactivator at the active site would be crucial for the design of an efficient reactivator.

  3. Reactivation of tabun-hAChE investigated by structurally analogous oximes and mutagenesis.

    PubMed

    Artursson, Elisabet; Akfur, Christine; Hörnberg, Andreas; Worek, Franz; Ekström, Fredrik

    2009-11-30

    The nerve agent tabun inhibits the essential enzyme acetylcholinesterase (AChE) by a rapid phosphoramidation of the catalytic serine residue. Oximes, such as K027 and HLö-7, can reactivate tabun-inhibited human AChE (tabun-hAChE) whereas the activity of their close structural analogue HI-6 is notably low. To investigate HI-6, K027 and HLö-7, residues lining the active-site gorge of hAChE were substituted and the effects on kinetic parameters for reactivation were determined. None of the mutants (Asp74Asn, Asp74Glu, Tyr124Phe, Tyr337Ala, Tyr337Phe, Phe338Val and Tyr341Ala) were able to facilitate HI-6-mediated reactivation of tabun-hAChE. In contrast, Tyr124Phe and Tyr337Phe induce a 2-2.5-fold enhancement of the bimolecular rate constant for K027 and HLö-7. The largest effects on the dissociation constant (3.5-fold increase) and rate constant (20-fold decrease) were observed for Tyr341Ala and Asp74Asn, respectively. These findings demonstrate the importance of residues located distant from the conjugate during the reactivation of tabun-hAChE.

  4. Molecular and Kinetic Properties of Two Acetylcholinesterases from the Western Honey Bee, Apis mellifera

    PubMed Central

    Kim, Young Ho; Cha, Deok Jea; Jung, Je Won; Kwon, Hyung Wook; Lee, Si Hyeock

    2012-01-01

    We investigated the molecular and kinetic properties of two acetylcholinesterases (AmAChE1 and AmAChE2) from the Western honey bee, Apis mellifera. Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Both AmAChEs exist as homodimers; the monomers are covalently connected via a disulfide bond under native conditions. However, AmAChE2 was associated with the cell membrane via the glycophosphatidylinositol anchor, while AmAChE1 was present as a soluble form. The two AmAChEs were functionally expressed with a baculovirus system. Kinetic analysis revealed that AmAChE2 has approximately 2,500-fold greater catalytic efficiency toward acetylthiocholine and butyrylthiocholine than AmAChE1, supporting the synaptic function of AmAChE2. In addition, AmAChE2 likely serves as the main target of the organophosphate (OP) and carbamate (CB) insecticides as judged by the lower IC50 values against AmAChE2 than against AmAChE1. When OP and CB insecticides were pre-incubated with a mixture of AmAChE1 and AmAChE2, a significant reduction in the inhibition of AmAChE2 was observed, suggesting a protective role of AmAChE1 against xenobiotics. Taken together, based on their tissue distribution pattern, molecular and kinetic properties, AmAChE2 plays a major role in synaptic transmission, while AmAChE1 has non-neuronal functions, including chemical defense. PMID:23144990

  5. Molecular and kinetic properties of two acetylcholinesterases from the western honey bee, Apis mellifera.

    PubMed

    Kim, Young Ho; Cha, Deok Jea; Jung, Je Won; Kwon, Hyung Wook; Lee, Si Hyeock

    2012-01-01

    We investigated the molecular and kinetic properties of two acetylcholinesterases (AmAChE1 and AmAChE2) from the Western honey bee, Apis mellifera. Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Both AmAChEs exist as homodimers; the monomers are covalently connected via a disulfide bond under native conditions. However, AmAChE2 was associated with the cell membrane via the glycophosphatidylinositol anchor, while AmAChE1 was present as a soluble form. The two AmAChEs were functionally expressed with a baculovirus system. Kinetic analysis revealed that AmAChE2 has approximately 2,500-fold greater catalytic efficiency toward acetylthiocholine and butyrylthiocholine than AmAChE1, supporting the synaptic function of AmAChE2. In addition, AmAChE2 likely serves as the main target of the organophosphate (OP) and carbamate (CB) insecticides as judged by the lower IC(50) values against AmAChE2 than against AmAChE1. When OP and CB insecticides were pre-incubated with a mixture of AmAChE1 and AmAChE2, a significant reduction in the inhibition of AmAChE2 was observed, suggesting a protective role of AmAChE1 against xenobiotics. Taken together, based on their tissue distribution pattern, molecular and kinetic properties, AmAChE2 plays a major role in synaptic transmission, while AmAChE1 has non-neuronal functions, including chemical defense.

  6. A Mechanism-based 3D-QSAR Approach for Classification and Prediction of Acetylcholinesterase Inhibitory Potency of Organophosphate and Carbamate Analogs

    EPA Science Inventory

    Organophosphate (OP) and carbamate esters can inhibit acetylcholinesterase (AChE) by binding covalently to a serine residue in the enzyme active site, and their inhibitory potency depends largely on affinity for the enzyme and the reactivity of the ester. Despite this understandi...

  7. Acetylcholinesterase-based organophosphate nerve agent sensing photonic crystal.

    PubMed

    Walker, Jeremy P; Asher, Sanford A

    2005-03-15

    We developed a polymerized crystalline colloidal array (PCCA) photonic crystal sensing material that senses the organophosphorus compound parathion at ultratrace concentrations in aqueous solutions. A periodic array of colloidal particles is embedded in a hydrogel network with a lattice spacing such that it Bragg diffracts visible light. The molecular recognition agent for the sensor is the enzyme acetylcholinesterase (AChE), which binds organophosphorus compounds irreversibly, creating an anionic phosphonyl species. This charged species creates a Donnan potential, which swells the hydrogel network, which increases the embedded particle array lattice spacing and causes a red-shift in the wavelength of light diffracted. The magnitude of the diffraction red-shift is proportional to the amount of bound parathion. These AChE-PCCAs act as dosimeters for parathion since it irreversibly binds. Parathion concentrations as low as 4.26 fM are easily detected.

  8. Targeting Acetylcholinesterase: Identification of Chemical Leads by High Throughput Screening, Structure Determination and Molecular Modeling

    PubMed Central

    Berg, Lotta; Andersson, C. David; Artursson, Elisabet; Hörnberg, Andreas; Tunemalm, Anna-Karin; Linusson, Anna; Ekström, Fredrik

    2011-01-01

    Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer's disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization

  9. Acetylcholinesterase Biosensors for Electrochemical Detection of Organophosphorus Compounds: A Review

    PubMed Central

    Dhull, Vikas; Gahlaut, Anjum; Dilbaghi, Neeraj

    2013-01-01

    The exponentially growing population, with limited resources, has exerted an intense pressure on the agriculture sector. In order to achieve high productivity the use of pesticide has increased up to many folds. These pesticides contain organophosphorus (OP) toxic compounds which interfere with the proper functioning of enzyme acetylcholinesterase (AChE) and finally affect the central nervous system (CNS). So, there is a need for routine, continuous, on spot detection of OP compounds which are the main limitations associated with conventional analytical methods. AChE based enzymatic biosensors have been reported by researchers as the most promising tool for analysis of pesticide level to control toxicity and for environment conservation. The present review summarises AChE based biosensors by discussing their characteristic features in terms of fabrication, detection limit, linearity range, time of incubation, and storage stability. Use of nanoparticles in recently reported fabrication strategies has improved the efficiency of biosensors to a great extent making them more reliable and robust. PMID:24383001

  10. Extracts and constituents of Leontopodium alpinum enhance cholinergic transmission: Brain ACh increasing and memory improving properties

    PubMed Central

    Hornick, Ariane; Schwaiger, Stefan; Rollinger, Judith M.; Vo, Nguyen Phung; Prast, Helmut; Stuppner, Hermann

    2012-01-01

    Leontopodium alpinum (‘Edelweiss’) was phytochemically investigated for constituents that might enhance cholinergic neurotransmission. The potency to increase synaptic availability of acetylcholine (ACh) in rat brain served as key property for the bioguided isolation of cholinergically active compounds using different chromatographic techniques. The dichlormethane (DCM) extract of the root, fractions and isolated constituents were injected i.c.v. and the effect on brain ACh was detected via the push–pull technique. The DCM extract enhanced extracellular ACh concentration in rat brain and inhibited acetylcholinesterase (AChE) in vitro. The extracellular level of brain ACh was significantly increased by the isolated sesquiterpenes, isocomene and 14-acetoxyisocomene, while silphiperfolene acetate and silphinene caused a small increasing tendency. Only silphiperfolene acetate showed in vitro AChE inhibitory activity, thus suggesting the other sesquiterpenes to stimulate cholinergic transmission by an alternative mechanism of action. Isocomene was further investigated with behavioural tasks in mice. It restored object recognition in scopolamine-impaired mice and showed nootropic effects in the T-maze alternation task in normal and scopolamine-treated mice. Additionally, this sesquiterpene reduced locomotor activity of untreated mice in the open field task, while the activity induced by scopolamine was abolished. The enhancement of synaptic availability of ACh, the promotion of alternation, and the amelioration of scopolamine-induced deficit are in accordance with a substance that amplifies cholinergic transmission. Whether the mechanism of action is inhibition of AChE or another pro-cholinergic property remains to be elucidated. Taken together, isocomene and related constituents of L. alpinum deserve further interest as potential antidementia agents in brain diseases associated with cholinergic deficits. PMID:18541221

  11. Cloning and expression of acetylcholinesterase from Bungarus fasciatus venom. A new type of cooh-terminal domain; involvement of a positively charged residue in the peripheral site.

    PubMed

    Cousin, X; Bon, S; Duval, N; Massoulié, J; Bon, C

    1996-06-21

    As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.

  12. Novel Human Butyrylcholinesterase Variants: Toward Organophosphonate Detoxication

    PubMed Central

    2015-01-01

    Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for stoichiometric binding and/or catalytic hydrolysis of organophosphates. Herein, we describe the use of a molecular evolution method to develop novel hBChE variants with increased resistance to stereochemically defined nerve agent model compounds of soman, sarin, and cyclosarin. Novel hBChE variants (Y332S, D340H, and Y332S/D340H) were identified with an increased resistance to nerve agent model compounds that retained robust intrinsic catalytic efficiency. Molecular dynamics simulations of these variants revealed insights into the mechanism by which these structural changes conferred nerve agent model compound resistance. PMID:24902043

  13. Alkaloid metabolite profiles by GC/MS and acetylcholinesterase inhibitory activities with binding-mode predictions of five Amaryllidaceae plants.

    PubMed

    Cortes, Natalie; Alvarez, Rafael; Osorio, Edison H; Alzate, Fernando; Berkov, Strahil; Osorio, Edison

    2015-01-01

    Acetylcholinesterase (AChE) enzymatic inhibition is an important target for the management of Alzheimer disease (AD) and AChE inhibitors are the mainstay drugs for its treatment. In order to discover new sources of potent AChE inhibitors, a combined strategy is presented based on AChE-inhibitory activity and chemical profiles by GC/MS, together with in silico studies. The combined strategy was applied on alkaloid extracts of five Amaryllidaceae species that grow in Colombia. Fifty-seven alkaloids were detected using GC/MS, and 21 of them were identified by comparing their mass-spectral fragmentation patterns with standard reference spectra in commercial and private library databases. The alkaloid extracts of Zephyranthes carinata exhibited a high level of inhibitory activity (IC50 = 5.97 ± 0.24 μg/mL). Molecular modeling, which was performed using the structures of some of the alkaloids present in this extract and the three-dimensional crystal structures of AChE derived from Torpedo californica, disclosed their binding configuration in the active site of this AChE. The results suggested that the alkaloids 3-epimacronine and lycoramine might be of interest for AChE inhibition. Although the galanthamine group is known for its potential utility in treating AD, the tazettine-type alkaloids should be evaluated to find more selective compounds of potential benefit for AD.

  14. Docking and molecular dynamics studies of peripheral site ligand-oximes as reactivators of sarin-inhibited human acetylcholinesterase.

    PubMed

    de Almeida, Joyce S F D; Cuya Guizado, Teobaldo R; Guimarães, Ana P; Ramalho, Teodorico C; Gonçalves, Arlan S; de Koning, Martijn C; França, Tanos C C

    2016-12-01

    In the present work, we performed docking and molecular dynamics simulations studies on two groups of long-tailored oximes designed as peripheral site binders of acetylcholinesterase (AChE) and potential penetrators on the blood brain barrier. Our studies permitted to determine how the tails anchor in the peripheral site of sarin-inhibited human AChE, and which aminoacids are important to their stabilization. Also the energy values obtained in the docking studies corroborated quite well with the experimental results obtained before for these oximes.

  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. Mutation in the human acetylcholinesterase-associated collagen gene, COLQ, is responsible for congenital myasthenic syndrome with end-plate acetylcholinesterase deficiency (Type Ic).

    PubMed Central

    Donger, C; Krejci, E; Serradell, A P; Eymard, B; Bon, S; Nicole, S; Chateau, D; Gary, F; Fardeau, M; Massoulié, J; Guicheney, P

    1998-01-01

    Congenital myasthenic syndrome (CMS) with end-plate acetylcholinesterase (AChE) deficiency is a rare autosomal recessive disease, recently classified as CMS type Ic (CMS-Ic). It is characterized by onset in childhood, generalized weakness increased by exertion, refractoriness to anticholinesterase drugs, and morphological abnormalities of the neuromuscular junctions (NMJs). The collagen-tailed form of AChE, which is normally concentrated at NMJs, is composed of catalytic tetramers associated with a specific collagen, COLQ. In CMS-Ic patients, these collagen-tailed forms are often absent. We studied a large family comprising 11 siblings, 6 of whom are affected by a mild form of CMS-Ic. The muscles of the patients contained collagen-tailed AChE. We first excluded the ACHE gene (7q22) as potential culprit, by linkage analysis; then we mapped COLQ to chromosome 3p24.2. By analyzing 3p24.2 markers located close to the gene, we found that the six affected patients were homozygous for an interval of 14 cM between D3S1597 and D3S2338. We determined the COLQ coding sequence and found that the patients present a homozygous missense mutation, Y431S, in the conserved C-terminal domain of COLQ. This mutation is thought to disturb the attachment of collagen-tailed AChE to the NMJ, thus constituting the first genetic defect causing CMS-Ic. PMID:9758617

  17. Effect of Calea serrata Less. n-hexane extract on acetylcholinesterase of larvae ticks and brain Wistar rats.

    PubMed

    Ribeiro, Vera Lucia Sardá; Vanzella, Cláudia; Moysés, Felipe dos Santos; Santos, Jaqueline Campiol Dos; Martins, João Ricardo Souza; von Poser, Gilsane Lino; Siqueira, Ionara Rodrigues

    2012-10-26

    Acetylcholinesterase (AChE), an enzyme that hydrolyses acetylcholine (ACh) at cholinergic synapses, is a target for pesticides and its inhibition by organophosphates leads to paralysis and death of arthropods. It has been demonstrated that the n-hexane extract of Calea serrata had acaricidal activity against larvae of Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus. The aim of the present study was to understand the mechanism of the acaricidal action of C. serrata n-hexane extract are specifically to investigate the in vitro anticholinesterase activity on larvae of R. microplus and in brain structures of male Wistar rats. The n-hexane extract significantly inhibited in vitro acetylcholinesterase activity in R. microplus larvae and rat brain structures. The results confirm that inhibition of acetylcholinesterase is a possible mechanism of action of hexane extract at C. serrata.

  18. How Is Acetylcholinesterase Phosphonylated by Soman? An Ab Initio QM/MM Molecular Dynamics Study

    PubMed Central

    2015-01-01

    Acetylcholinesterase (AChE) is a crucial enzyme in the cholinergic nerve system that hydrolyzes acetylcholine (ACh) and terminates synaptic signals by reducing the effective concentration of ACh in the synaptic clefts. Organophosphate compounds irreversibly inhibit AChEs, leading to irreparable damage to nerve cells. By employing Born–Oppenheimer ab initio QM/MM molecular dynamics simulations with umbrella sampling, a state-of-the-art approach to simulate enzyme reactions, we have characterized the covalent inhibition mechanism between AChE and the nerve toxin soman and determined its free energy profile for the first time. Our results indicate that phosphonylation of the catalytic serine by soman employs an addition–elimination mechanism, which is highly associative and stepwise: in the initial addition step, which is also rate-limiting, His440 acts as a general base to facilitate the nucleophilic attack of Ser200 on the soman’s phosphorus atom to form a trigonal bipyrimidal pentacovalent intermediate; in the subsequent elimination step, Try121 of the catalytic gorge stabilizes the leaving fluorine atom prior to its dissociation from the active site. Together with our previous characterization of the aging mechanism of soman inhibited AChE, our simulations have revealed detailed molecular mechanistic insights into the damaging function of the nerve agent soman. PMID:24786171

  19. Effect of carbaryl (carbamate insecticide) on acetylcholinesterase activity of two strains of Daphnia magna (Crustacea, Cladocera).

    PubMed

    Toumi, Hela; Bejaoui, Mustapha; Touaylia, Samir; Burga Perez, Karen F; Ferard, Jean François

    2016-11-01

    The present study was designed to investigate the effect of carbaryl (carbamate insecticide) on the acetylcholinesterase activity in two strains (same clone A) of the crustacean cladoceran Daphnia magna. Four carbaryl concentrations (0.4, 0.9, 1.8 and 3.7 µg L(-1)) were compared against control AChE activity. Our results showed that after 48 h of carbaryl exposure, all treatments induced a significant decrease of AChE activities whatever the two considered strains. However, different responses were registered in terms of lowest observed effect concentrations (LOEC: 0.4 µg L(-1) for strain 1 and 0.9 µg L(-1) for strains 2) revealing differences in sensitivity among the two tested strains of D. magna. These results suggest that after carbaryl exposure, the AChE activity responses can be also used as a biomarker of susceptibility. Moreover, our results show that strain1 is less sensitive than strain 2 in terms of IC50-48 h of AChE activity. Comparing the EC50-48 h of standard ecotoxicity test and IC50-48 h of AChE inhibition, there is the same order of sensitivity with both strains.

  20. Acetylcholinesterase inhibitory, antioxidant and phytochemical properties of selected medicinal plants of the Lamiaceae family.

    PubMed

    Vladimir-Knežević, Sanda; Blažeković, Biljana; Kindl, Marija; Vladić, Jelena; Lower-Nedza, Agnieszka D; Brantner, Adelheid H

    2014-01-09

    The present study aimed to evaluate acetylcholinesterase (AChE) inhibitory and antioxidant activities of Lamiaceae medicinal plants growing wild in Croatia. Using Ellman's colorimetric assay all tested ethanolic extracts and their hydroxycinnamic acid constituents demonstrated in vitro AChE inhibitory properties in a dose dependent manner. The extracts of Mentha x piperita, M. longifolia, Salvia officinalis, Satureja montana, Teucrium arduini, T. chamaedrys, T. montanum, T. polium and Thymus vulgaris at 1 mg/mL showed strong inhibitory activity against AChE. The antioxidant potential of the investigated Lamiaceae species was assessed by DPPH• scavenging activity and total antioxidant capacity assays, in comparison with hydroxycinnamic acids and trolox. The extracts differed greatly in their total hydroxycinnamic derivatives content, determined spectrophotometrically. Rosmarinic acid was found to be the predominant constituent in most of the investigated medicinal plants (by RP-HPLC) and had a substantial influence on their AChE inhibitory and antioxidant properties, with the exception of Teucrium species. These findings indicate that Lamiaceae species are a rich source of various natural AChE inhibitors and antioxidants that could be useful in the prevention and treatment of Alzheimer's and other related diseases.

  1. Oxidized low density lipoprotein increases acetylcholinesterase activity correlating with reactive oxygen species production.

    PubMed

    Yamchuen, Panit; Aimjongjun, Sathid; Limpeanchob, Nanteetip

    2014-12-01

    Hyperlipidemia, low density lipoproteins (LDL) and their oxidized forms, and oxidative stress are suspected to be a key combination in the onset of AD and acetylcholinesterase (AChE) plays a part in this pathology. The present study aimed to link these parameters using differentiated SH-SY5Y human neuroblastoma cells in culture. Both mildly and fully oxidized human LDL (mox- and fox-LDL), but not native (non-oxidized) LDL were cytotoxic in dose- and time-dependent patterns and this was accompanied by an increased production of intracellular reactive oxygen species (ROS). Oxidized LDL (10-200 μg/mL) augmented AChE activity after 4 and 24h treatments, respectively while the native LDL was without effect. The increased AChE with oxidized LDLs was accompanied by a proportionate increase in intracellular ROS formation (R=0.904). These findings support the notion that oxidized LDLs are cytotoxic and that their action on AChE may reduce central cholinergic transmission in AD and affirm AChE as a continued rational for anticholinesterase therapy but in conjunction with antioxidant/antihyperlipidemic cotreatments.

  2. Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet.

    PubMed

    Martín-Biosca, Yolanda; Asensi-Bernardi, Lucia; Villanueva-Camañas, Rosa M; Sagrado, Salvador; Medina-Hernández, Maria J

    2009-05-01

    In this study the development of a procedure based on capillary electrophoresis after enzymatic reaction at capillary inlet methodology for the screening and in vitro evaluation of the biological activity of acetylcholinesterase (AChE) inhibitors is presented. The progress of the enzymatic reaction of the hydrolysis of acetylthiocholine at pH 8 in the presence of AChE and the inhibitor studied is determined by measuring at 230 nm the peak area of the reaction product thiocholine (TCh). In the method employed the capillary was first filled with 30 mM borate-phosphate buffer (pH 8.0) and subsequently, plugs of: (i) water, (ii) AChE solution, (iii) substrate solution with or without inhibitor, (iv) AChE solution, and (v) water, were hydrodynamically injected into the capillary, and were allowed to stand (and react) during a waiting period of 2 min. The applicability of the proposed methodology to estimate different kinetic parameters of interest such as inhibition constants K(i), identification of inhibitory action mechanism and IC(50), is evaluated using compounds with known activity, tacrine edrophonium, and neostigmine. The results obtained are compared with bibliographic values and confirm the effectiveness of the methodology proposed. Finally a method for AChE Inhibitor screening is proposed.

  3. Cholinesterase inhibitors: xanthostigmine derivatives blocking the acetylcholinesterase-induced beta-amyloid aggregation.

    PubMed

    Belluti, Federica; Rampa, Angela; Piazzi, Lorna; Bisi, Alessandra; Gobbi, Silvia; Bartolini, Manuela; Andrisano, Vincenza; Cavalli, Andrea; Recanatini, Maurizio; Valenti, Piero

    2005-06-30

    In continuing research that led us to identify a new class of carbamate derivatives acting as potent (Rampa et al. J. Med. Chem. 1998, 41, 3976) and long-lasting (Rampa et al. J. Med. Chem. 2001, 44, 3810) acetylcholinesterase (AChE) inhibitors, we obtained some analogues able to simultaneously block both the catalytic and the beta-amyloid (Abeta) proaggregatory activities of AChE. The key feature of these derivatives is a 2-arylidenebenzocycloalkanone moiety that provides the ability to bind at the AChE peripheral site responsible for promoting the Abeta aggregation. The new carbamates were tested in vitro for the inhibition of both cholinesterases and also for the ability to prevent the AChE-induced Abeta aggregation. All of the compounds had AChE IC(50) values in the nanomolar range and showed the ability to block the AChE-induced Abeta aggregation, thus supporting the feasibility of this new strategy in the search of compounds for the treatment of Alzheimer's disease.

  4. Novel Selective and Irreversible Mosquito Acetylcholinesterase Inhibitors for Controlling Malaria and Other Mosquito-Borne Diseases

    NASA Astrophysics Data System (ADS)

    Dou, Dengfeng; Park, Jewn Giew; Rana, Sandeep; Madden, Benjamin J.; Jiang, Haobo; Pang, Yuan-Ping

    2013-01-01

    We reported previously that insect acetylcholinesterases (AChEs) could be selectively and irreversibly inhibited by methanethiosulfonates presumably through conjugation to an insect-specific cysteine in these enzymes. However, no direct proof for the conjugation has been published to date, and doubts remain about whether such cysteine-targeting inhibitors have desirable kinetic properties for insecticide use. Here we report mass spectrometric proof of the conjugation and new chemicals that irreversibly inhibited African malaria mosquito AChE with bimolecular inhibition rate constants (kinact/KI) of 3,604-458,597 M-1sec-1 but spared human AChE. In comparison, the insecticide paraoxon irreversibly inhibited mosquito and human AChEs with kinact/KI values of 1,915 and 1,507 M-1sec-1, respectively, under the same assay conditions. These results further support our hypothesis that the insect-specific AChE cysteine is a unique and unexplored target to develop new insecticides with reduced insecticide resistance and low toxicity to mammals, fish, and birds for the control of mosquito-borne diseases.

  5. A computational insight into acetylcholinesterase inhibitory activity of a new lichen depsidone.

    PubMed

    Ece, Abdulilah; Pejin, Boris

    2015-01-01

    Acetylcholinesterase (AChE) inhibitors are yet the best drugs currently available for the management of Alzheimer's disease. The recent phytochemical investigation has led to the isolation of a new depsidone 1 with moderate AChE activity (1 μg). This work was focused on its electronic properties analysed using commercially available programs. Both the active depsidone molecule 1 and galanthamine showed to have higher HOMO energies than the inactive depsidones 2-4, isolated from the same lichen species. However, the amino depsidone derivative 7, whose structure was proposed using computational approaches, is expected to be more active AChE inhibitor than the depsidone 1, due to the improved HOMO energy value. In addition, the molecular docking study indicated that the compound 7 has ability to make the well-known interactions of potent AChE inhibitors with the enzyme active site. The data presented herein support the design of novel AChE inhibitors based on the depsidone scaffold.

  6. Amaryllidaceae alkaloids with new framework types from Zephyranthes candida as potent acetylcholinesterase inhibitors.

    PubMed

    Zhan, Guanqun; Liu, Junjun; Zhou, Junfei; Sun, Bin; Aisa, Haji Akber; Yao, Guangmin

    2017-02-15

    Three new Amaryllidaceae alkaloids, named zephycandidines I-III (1-3), were isolated from Zephyranthes candida. The structures of 1-3 were elucidated by spectroscopic analyses including HRESIMS, (1)H NMR, (13)C NMR, DEPT, HSQC, (1)H-(1)H COSY, HMBC, ROESY, and electronic circular dichroism (ECD), as well as ECD calculation. The absolute configuration of 1 was finally established by single crystal X-ray diffraction using Cu Kα radiation. Zephycandidines I (1) and III (3) with new framework types represent the first example of 7-phenyl-hexahydroindole and 5,2'-dimethyl-biphenyl-2-ylamine alkaloids, respectively, and their plausible biosynthetic pathway are proposed. Zephycandidine II (2) is the first C3a-phenyl-hexahydroindole type alkaloid isolated from the genus of Zephyranthes. These new alkaloids 1-3 were evaluated for their acetylcholinesterase (AChE) inhibitory activities, and 3 showed potent AChE inhibitory activity with an IC50 value of 8.82 μM, suggesting that the framework of 5,2'-dimethyl-biphenyl-2-ylamine in 3 may be a potential group for the AChE inhibitory activity. The docking studies of 1-3 and galanthamine with AChE revealed that interactions with W286 and Y337 are necessary for the AChE inhibitory activity.

  7. Variations in Acetylcholinesterase Activity within Human Cortical Pyramidal Neurons Across Age and Cognitive Trajectories.

    PubMed

    Janeczek, Monica; Gefen, Tamar; Samimi, Mehrnoosh; Kim, Garam; Weintraub, Sandra; Bigio, Eileen; Rogalski, Emily; Mesulam, M-Marsel; Geula, Changiz

    2017-03-01

    We described an extensive network of cortical pyramidal neurons in the human brain with abundant acetylcholinesterase (AChE) activity. Emergence of these neurons during childhood/adolescence, attainment of highest density in early adulthood, and virtual absence in other species led us to hypothesize involvement of AChE within these neurons in higher cortical functions. The current study quantified the density and staining intensity of these neurons using histochemical procedures. Few faintly stained AChE-positive cortical pyramidal neurons were observed in children/adolescents. These neurons attained their highest density and staining intensity in young adulthood. Compared with the young adult group, brains of cognitively normal elderly displayed no significant change in numerical density but a significant decrease in staining intensity of AChE-positive cortical pyramidal neurons. Brains of elderly above age 80 with unusually preserved memory performance (SuperAgers) showed significantly lower staining intensity and density of these neurons when compared with same-age peers. Conceivably, low levels of AChE activity could enhance the impact of acetylcholine on pyramidal neurons to counterbalance other involutional factors that mediate the decline of memory capacity during average aging. We cannot yet tell if elderly with superior memory capacity have constitutively low neuronal AChE levels or if this feature reflects adaptive neuroplasticity.

  8. Brain acetylcholinesterase diurnal variations during the rapid development of tolerance to the hypothermic effect of ethanol

    SciTech Connect

    Wang, O.; Soliman, K.F.A. )

    1991-03-11

    Male Sprague-Dawley rats maintained under controlled environmental conditions were used. Acetylcholinesterase (AChE) activity was determined in the cerebral cortex, midbrain, hypothalamus, hippocampus, cerebellum, pons and medulla oblongata of saline control and ethanol-treated rats, either after a single dose at 06:0 or 18:00h, or after a second dose administered 24 hrs later at the same time scheduled. Results of this experiment indicate that repeated administration with ethanol was associated with the rapid development of tolerance to the hypothermic action of ethanol. A single injection of ethanol at 0600h resulted in a significant decrease in AChE activity in the hypothalamus, medulla, cerebellum, hippocampus and the cortex. However, ethanol administration at 18.00h was associated with significant increases in AChE activity in the same brain regions. The repeated administration of ethanol at 06.00h was associated with tolerance in AChE response to ethanol in the hypothalamus and hippocampus. However, there was no tolerance development in AChE activity in brain regions when ethanol was administered at 18.00h. The results indicate that chronotolerance to ethanol might be related to the brain cholinergic system.

  9. Influence of Pb sup ++ on expression of acetylcholinesterase and dihydropyridine receptors in avian skeletal muscle

    SciTech Connect

    Luo, Zhigang; Berman, H.A. )

    1991-03-11

    These studies examine the influence of inorganic lead on expression of dihydropyridine receptors and molecular forms of acetylcholinesterase (AchE) in primary cultures of avian skeletal muscles. Treatment of avian muscle cultures with Pb{sup ++} in the concentration range 0-100 {mu}M caused graded reductions in the amounts of dihydropyridine receptors. These actions required at least 12 hours exposure to Pb{sup ++}, occurred without change in receptor affinity, and caused no discernible reduction in contractile activity of the cultured fibers. Under similar conditions treatment with Pb{sup ++} caused comparable reductions in the presence of AchE; in these cases the principal actions were observed as reductions in 7S AchE, an intracellular form of the enzyme. Since these actions are not observed in neural retina, they are concluded to be specific for skeletal muscle. The heterologous down regulation of both dihydropyridine receptors and specific intracellular forms of AchE indicates a distinct linkage between regulation of voltage-dependent calcium channels and AchE, as well as a pleiotropic activity of Pb{sup ++} on protein biosynthesis in general. In addition, these relationships suggest a novel mechanism through which Pb{sup ++} exerts its chronic mode of toxicity.

  10. Isolating a Cell Maximally Secreting Acetylcholinesterase

    DTIC Science & Technology

    1987-05-12

    production of AChE via gene duplication 12 4. Molecular cloning of the AChE gene 12 5. Production of AChE 12 6. Characterization of the Final Product... Molecular cloning of the AChE gene. From overproducing cell lines which had been developed as described in the previous section (3), the AChE gene was...to have been isolated by molecular cloning . 5. Provision of AChE. At the end of the contract, at least 100 milligrams of human AChE was to have been

  11. Exploration of the susceptibility of AChE from the poultry red mite Dermanyssus gallinae (Acari: Mesostigmata) to organophosphates in field isolates from France.

    PubMed

    Roy, Lise; Chauve, Claude; Delaporte, Jean; Inizan, Gilbert; Buronfosse, Thierry

    2009-06-01

    The red fowl mite Dermanyssus gallinae (De Geer, 1778) is a hematophagous mite species, which is very commonly found in layer facilities in Europe. The economic and animal health impact of this parasite is quite important. In laying hen houses, organophosphates are almost the only legally usable chemicals. Detecting a target resistance can be useful in order to limit the emergence of resistant populations. The acetylcholinesterase (AChE) activity and the enzyme sensitivity to paraoxon was investigated in 39 field samples and compared to a susceptible reference strain (SSK). Insensitivity factor values (expressed as IC50 ratio) obtained from field isolates compared to SSK revealed some polymorphism but not exceeding a 6-fold difference. The kinetic characteristics of AChE from some field samples showed some difference in KM values for acetylthiocholine and inhibition kinetics performed with diethyl paraoxon exhibited a 5.5-fold difference in the bimolecular rate constant in one field isolate. Taken together, these data suggested that differences in AChE susceptibility to organophosphates may exist in D. gallinae but no resistant population was found.

  12. Design of multi-target compounds as AChE, BACE1, and amyloid-β(1-42) oligomerization inhibitors: in silico and in vitro studies.

    PubMed

    Hernández-Rodríguez, Maricarmen; Correa-Basurto, José; Martínez-Ramos, Federico; Padilla-Martínez, Itzia Irene; Benítez-Cardoza, Claudia G; Mera-Jiménez, Elvia; Rosales-Hernández, Martha Cecilia

    2014-01-01

    Despite great efforts to develop new therapeutic strategies against Alzheimer's disease (AD), the acetylcholinesterase inhibitors (AChEIs): donepezil, rivastigmine, and galantamine, have been used only as a palliative therapeutic approach. However, the pathogenesis of AD includes several factors such as cholinergic hypothesis, amyloid-β (Aβ) aggregation, and oxidative stress. For this reason, the design of compounds that target the genesis and progression of AD could offer a therapeutic benefit. We have designed a set of compounds (M-1 to M-5) with pharmacophore moieties to inhibit the release, aggregation, or toxicity of Aβ, act as AChEIs and have antioxidant properties. Once the compounds were designed, we analyzed their physicochemical parameters and performed docking studies to determine their affinity values for AChE, β-site amyloid-protein precursor cleaving enzyme 1 (BACE1), and the Aβ monomer. The best ligands, M-1 and M-4, were then synthesized, chemically characterized, and evaluated in vitro. The in vitro studies showed that these compounds inhibit AChE (M-1 Ki = 0.12 and M-4 Ki = 0.17 μM) and BACE1 (M-1 IC50 = 15.1 and M-4 IC50 = 15.4 nM). They also inhibit Aβ oligomerization and exhibit antioxidant activity. In addition, these compounds showed low cytotoxicity in microglial cells. For these reasons, they are promising for future use as drugs in AD mice transgenic models.

  13. Acetylcholinesterase inhibition in the threeridge mussel (Amblema plicata) by chlorpyrifos: implications for biomonitoring

    USGS Publications Warehouse

    Doran, W.J.; Cope, W.G.; Rada, R.G.; Sandheinrich, M.B.

    2001-01-01

    The effects of chlorpyrifos, an organophosphorus insecticide, were examined on the activity of the nervous system enzyme acetylcholinesterase (AChE) in the threeridge mussel Amblema plicata in a 24-day laboratory test. Thirty-six mussels in each of seven treatments (18 mussels per duplicate) were exposed to chlorpyrifos (0.1, 0.2, 0.3, 0.6, and 1.2 mg/L), a solvent (acetone), and a solvent-free (well water) control for 12, 24, or 96 h. The activity of AChE was measured in the anterior adductor muscle of eight mussels from each treatment after exposure. To assess potential latent effects, six mussels from each treatment were removed after 24 h of exposure and transferred to untreated water for a 21-day holding period; AChE activity was measured on three mussels from each treatment at 7 and 21 days of the holding period. The activity of AChE in chlorpyrifos-exposed mussels did not differ from controls after 12 or 24 h of exposure (t- test, P>0.05), but was significantly less than controls after 96 h (t- test, P=0.01). AChE activity did not vary among mussels at 24 h of exposure (i.e., Day 0 of holding period) and those at Day 7 and Day 21 of the holding period. Overall changes in AChE activity of mussels during the test were unrelated to individual chlorpyrifos concentrations and exposure times (repeated measure ANOVA; (P=0.06). A power analysis revealed that the sample size must be increased from 2 to 5 replicates (8 to 20 mussels per time interval and test concentration) to increase the probability of detecting significant differences in AChE activity. This calculated increase in sample size has potential implications for future biomonitoring studies with chlorpyrifos and unionid mussels.

  14. Bis-quaternary oximes amplify the effectiveness of acetylcholinesterase to detoxify organophosphorus compounds

    SciTech Connect

    Caranto, G.R.; Waibel, K.H.; Asher, J.M.; Larrison, R.W.; Brecht, K.M.

    1993-05-13

    Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase (FBS AChE) provides complete protection against 5 LD(50), of organophosphate (OP) without any signs of toxicity or performance decrements as measured by serial probe recognition tests or primate equilibrium platform performance (7,8). Although such use of enzyme as a single pretreatment drug for OP toxicity is sufficient to provide complete protection, a relatively large (stoichiometric) amount of enzyme was required in vivo to neutralize OP. To improve the efficacy of ChEs as pretreatment drugs, we have developed an approach in which the catalytic activity of OP-inhibited FBS AChE was rapidly and continuously restored, thus detoxifying the OP and minimizing enzyme aging by having sufficient amounts of appropriate oxime present. The efficacy of FBS AChE to detoxify several OPs was amplified by addition of bisquaternary oximes, particularly HI-6. When mice were pretreated with sufficient amounts of FBS AChE and HI-6 and challenged with repeated doses of sarin, the OP was continuously detoxified so long as the molar concentration of the sarin dose was less than the molar concentration of AChE in circulation. The in vitro experiments showed that the stoichiometry of sarin:FBS AChE was higher than 3200:1 and in vivo stoichiometry with mice was as high as 57:1. Addition of HI-6 to FBS AChE as a pretreatment drug amplified the efficacy of enzyme as a scavenger of nerve agents.

  15. Synthesis and acetylcholinesterase inhibitory activity of polyhydroxylated sulfated steroids: structure/activity studies.

    PubMed

    Richmond, Victoria; Murray, Ana P; Maier, Marta S

    2013-11-01

    Disulfated and trisulfated steroids have been synthesized from cholesterol and their acetylcholinesterase inhibitory activity has been evaluated. In our studies we have found that the activity was not only dependent on the location of the sulfate groups but on their configurations. 2β,3α,6α-trihydroxy-5α-cholestan-6-one trisulfate (18) was the most active steroid with an IC50 value of 15.48 μM comparable to that of 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (1). Both compounds were found to be less active than the reference compound eserine. The butyrylcholinesterase activity of 1 and 18 was one magnitude lower than that against acetylcholinesterase revealing a selective inhibitor profile.

  16. Positive cooperative regulation of double binding sites for human acetylcholinesterase.

    PubMed

    Liu, Hao; Ye, Wei; Chen, Hai-Feng

    2016-10-25

    Acetylcholinesterase is a potent enzyme that regulates neurotransmission by rapidly hydrolyzing the neurotransmitter acetylcholine in synapses of the nervous system. As drug target of anti-AD, it has catalytic and peripheral anionic sites. However, the regulation relation between these two sites is unclear. Therefore, we constructed dynamics fluctuation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in double-site system (hAChE/TZ5) is distinctly different from that in the free state and single-site systems (hAChE/huprine and hAChE/1YL). The community network analysis indicates that the information freely transfers from the peripheral anionic site to the catalytic active site in hAChE/TZ5. Furthermore, the binding free energy between the inhibitor and hAChE for hAChE/TZ5 is significantly lower than of either hAChE/huprine or hAChE/1YL. Thus, a hypothesis of 'positive cooperative regulation' is proposed for the regulation of double binding sites and further confirmed by the weakening and mutation community analyses. Finally, one possible cooperative regulation pathway of W86-TZ5-W286 was identified based on the shortest path algorithm and was confirmed by the network perturbation analysis. Interestingly, the regulation pathway for single-site systems is significantly different from that of dual-site system. The process targeting on the shortest pathway can better regulate the hydrolyzing the neurotransmitter acetylcholine and significantly inhibit the aggregation of Aβ amyloid.

  17. Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

    PubMed Central

    Bourne, Yves; Kolb, Hartmuth C.; Radić, Zoran; Sharpless, K. Barry; Taylor, Palmer; Marchot, Pascale

    2004-01-01

    The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1- and anti1-mouse AChE complexes at 2.45- to 2.65-Å resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template. PMID:14757816

  18. Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation.

    PubMed

    Bourne, Yves; Kolb, Hartmuth C; Radić, Zoran; Sharpless, K Barry; Taylor, Palmer; Marchot, Pascale

    2004-02-10

    The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1- and anti1-mouse AChE complexes at 2.45- to 2.65-A resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.

  19. Monoquaternary pyridinium salts with modified side chain-synthesis and evaluation on model of tabun- and paraoxon-inhibited acetylcholinesterase.

    PubMed

    Musilek, Kamil; Kucera, Jiri; Jun, Daniel; Dohnal, Vlastimil; Opletalova, Veronika; Kuca, Kamil

    2008-09-01

    Acetylcholinesterase reactivators are crucial antidotes for the treatment of organophosphate intoxication. Eighteen monoquaternary reactivators of acetylcholinesterase with modified side chain were developed in an effort to extend the properties of pralidoxime. The known reactivators (pralidoxime, HI-6, obidoxime, trimedoxime, methoxime) and the prepared compounds were tested in vitro on a model of tabun- and paraoxon-inhibited AChE. Monoquaternary reactivators were not able to exceed the best known compounds for tabun poisoning, but some of them did show reactivation better or comparable with pralidoxime for paraoxon poisoning. However, extensive differences were found by a SAR study for various side chains on the non-oxime part of the reactivator molecule.

  20. Fucoxanthin, a Marine Carotenoid, Reverses Scopolamine-Induced Cognitive Impairments in Mice and Inhibits Acetylcholinesterase in Vitro.

    PubMed

    Lin, Jiajia; Huang, Ling; Yu, Jie; Xiang, Siying; Wang, Jialing; Zhang, Jinrong; Yan, Xiaojun; Cui, Wei; He, Shan; Wang, Qinwen

    2016-03-25

    Fucoxanthin, a natural carotenoid abundant in edible brown seaweeds, has been shown to possess anti-cancer, anti-oxidant, anti-obesity and anti-diabetic effects. In this study, we report for the first time that fucoxanthin effectively protects against scopolamine-induced cognitive impairments in mice. In addition, fucoxanthin significantly reversed the scopolamine-induced increase of acetylcholinesterase (AChE) activity and decreased both choline acetyltransferase activity and brain-derived neurotrophic factor (BDNF) expression. Using an in vitro AChE activity assay, we discovered that fucoxanthin directly inhibits AChE with an IC50 value of 81.2 μM. Molecular docking analysis suggests that fucoxanthin likely interacts with the peripheral anionic site within AChE, which is in accordance with enzymatic activity results showing that fucoxanthin inhibits AChE in a non-competitive manner. Based on our current findings, we anticipate that fucoxanthin might exhibit great therapeutic efficacy for the treatment of Alzheimer's disease by acting on multiple targets, including inhibiting AChE and increasing BDNF expression.

  1. Effect of different buffers on kinetic properties of human acetylcholinesterase and the interaction with organophosphates and oximes.

    PubMed

    Wille, T; Thiermann, H; Worek, F

    2011-03-01

    Acetylcholinesterase (AChE) is the primary target of organophosphorus compounds (OP). The investigation into interactions between AChE, OP and oximes in vitro may be affected by the experimental conditions, e.g. by the buffer system. Hence, it was tempting to investigate the Michaelis-Menten kinetics and the inhibition and reactivation kinetics of paraoxon-ethyl, sarin, soman and VX in the presence of phosphate, MOPS, Tyrode and TRIS buffer with human AChE. Compared to phosphate buffer, the inhibition and reactivation kinetics of human erythrocyte AChE were markedly changed by TRIS and in part by MOPS, whereas Tyrode showed similar results to phosphate buffer. These results indicate an effect of the tested buffers on the properties of AChE, and an interaction between OP and oximes has to be considered for the design of in vitro studies and may impair the comparison of data from different laboratories. In view of the comparability of human in vitro kinetic data determined with phosphate buffer with data from human OP poisoning, it seems to be a suitable buffer for the investigation into interactions between AChE, OP and oximes.

  2. Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro

    SciTech Connect

    Herkert, N.M.; Schulz, S.; Wille, T.; Thiermann, H.; Hatz, R.A.; Worek, F.

    2011-05-15

    Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before or after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by decarbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.

  3. Fucoxanthin, a Marine Carotenoid, Reverses Scopolamine-Induced Cognitive Impairments in Mice and Inhibits Acetylcholinesterase in Vitro

    PubMed Central

    Lin, Jiajia; Huang, Ling; Yu, Jie; Xiang, Siying; Wang, Jialing; Zhang, Jinrong; Yan, Xiaojun; Cui, Wei; He, Shan; Wang, Qinwen

    2016-01-01

    Fucoxanthin, a natural carotenoid abundant in edible brown seaweeds, has been shown to possess anti-cancer, anti-oxidant, anti-obesity and anti-diabetic effects. In this study, we report for the first time that fucoxanthin effectively protects against scopolamine-induced cognitive impairments in mice. In addition, fucoxanthin significantly reversed the scopolamine-induced increase of acetylcholinesterase (AChE) activity and decreased both choline acetyltransferase activity and brain-derived neurotrophic factor (BDNF) expression. Using an in vitro AChE activity assay, we discovered that fucoxanthin directly inhibits AChE with an IC50 value of 81.2 μM. Molecular docking analysis suggests that fucoxanthin likely interacts with the peripheral anionic site within AChE, which is in accordance with enzymatic activity results showing that fucoxanthin inhibits AChE in a non-competitive manner. Based on our current findings, we anticipate that fucoxanthin might exhibit great therapeutic efficacy for the treatment of Alzheimer’s disease by acting on multiple targets, including inhibiting AChE and increasing BDNF expression. PMID:27023569

  4. Molecular, biochemical and histochemical characterization of two acetylcholinesterase cDNAs from the German cockroach Blattella germanica.

    PubMed

    Kim, J I; Jung, C S; Koh, Y H; Lee, S H

    2006-08-01

    Full length cDNAs encoding two acetylcholinesterases (AChEs; Bgace1 and Bgace2) were cloned and characterized from the German cockroach, Blattella germanica. Sequence analyses showed that both genes possess all the typical features of ace, and that Bgace1 is orthologous to the insect ace1 whereas Bgace2 is to the insect ace2. Transcript level of Bgace1 was significantly higher (c. 10 fold) than that of Bgace2 in all 11 tissues examined, suggesting that Bgace1 likely encodes a predominant AChE. Multiple AChE bands were identified by native polyacrylamide gel electrophoresis and isoelectricfocusing from various tissue preparations, among which ganglia produced distinct two major and two minor AChE bands, indicative of the presence of at least two active AChEs. B. germanica AChEs appeared to be mainly localized in the central nervous system as demonstrated by histochemical activity staining, together with quantitative analysis of Bgace transcripts. Fluorescence in situ hybridization of the 1st thoracic ganglion confirmed that Bgace1 is predominantly transcribed and further showed that its transcript is found in almost entire region of inter or motor neurones including the cell bodies and axonal/dendritic branches. Bgace2 transcript is found only in the subset of neurones, particularly in the cell body. In addition, certain neurones were observed to express Bgace1 only.

  5. Elaborate ligand-based modeling coupled with QSAR analysis and in silico screening reveal new potent acetylcholinesterase inhibitors

    NASA Astrophysics Data System (ADS)

    Abuhamdah, Sawsan; Habash, Maha; Taha, Mutasem O.

    2013-12-01

    Inhibition of the enzyme acetylcholinesterase (AChE) has been shown to alleviate neurodegenerative diseases prompting several attempts to discover and optimize new AChE inhibitors. In this direction, we explored the pharmacophoric space of 85 AChE inhibitors to identify high quality pharmacophores. Subsequently, we implemented genetic algorithm-based quantitative structure-activity relationship (QSAR) modeling to select optimal combination of pharmacophoric models and 2D physicochemical descriptors capable of explaining bioactivity variation among training compounds ( {{r}}^{ 2}_{ 6 8} = 0. 9 4 , F-statistic = 125.8, {{r}}^{ 2}_{{LOO}} { = 0} . 9 2 , {{r}}^{ 2}_{{PRESS}} against 17 external test inhibitors = 0.84). Two orthogonal pharmacophores emerged in the QSAR equation suggesting the existence of at least two binding modes accessible to ligands within AChE binding pocket. The successful pharmacophores were comparable with crystallographically resolved AChE binding pocket. We employed the pharmacophoric models and associated QSAR equation to screen the national cancer institute list of compounds. Twenty-four low micromolar AChE inhibitors were identified. The most potent gave IC50 value of 1.0 μM.

  6. Acetylcholinesterase inhibition reveals endogenous nicotinic modulation of glutamate inputs to CA1 stratum radiatum interneurons in hippocampal slices.

    PubMed

    Alkondon, Manickavasagom; Albuquerque, Edson X; Pereira, Edna F R

    2013-05-01

    The involvement of brain nicotinic acetylcholine receptors (nAChRs) in the neurotoxicological effects of soman, a potent acetylcholinesterase (AChE) inhibitor and a chemical warfare agent, is not clear. This is partly due to a poor understanding of the role of AChE in brain nAChR-mediated functions. To test the hypothesis that AChE inhibition builds sufficient acetylcholine (ACh) in the brain and facilitates nAChR-dependent glutamate transmission, we used whole-cell patch-clamp technique to record spontaneous glutamate excitatory postsynaptic currents (EPSCs) from CA1 stratum radiatum interneurons (SRI) in hippocampal slices. First, the frequency, amplitude and kinetics of EPSCs recorded from slices of control guinea pigs were compared to those recorded from slices of guinea pigs after a single injection of the irreversible AChE inhibitor soman (25.2μg/kg, s.c.). Second, EPSCs were recorded from rat hippocampal slices before and after their superfusion with the reversible AChE inhibitor donepezil (100nM). The frequency of EPSCs was significantly higher in slices taken from guinea pigs 24h but not 7 days after the soman injection than in slices from control animals. In 52% of the rat hippocampal slices tested, bath application of donepezil increased the frequency of EPSCs. Further, exposure to donepezil increased both burst-like and large-amplitude EPSCs, and increased the proportion of short (20-100ms) inter-event intervals. Donepezil's effects were suppressed significantly in presence of 10μM mecamylamine or 10nM methyllycaconitine. These results support the concept that AChE inhibition is able to recruit nAChR-dependent glutamate transmission in the hippocampus and such a mechanism can contribute to the acute neurotoxicological actions of soman.

  7. In silico pharmacophore model for tabun-inhibited acetylcholinesterase reactivators: a study of their stereoelectronic properties.

    PubMed

    Bhattacharjee, Apurba K; Kuca, Kamil; Musilek, Kamil; Gordon, Richard K

    2010-01-01

    Organophosphorus (OP) nerve agents that inhibit acetylcholinesterase (AChE; EC 3.1.1.7) function in the nervous system, causing acute intoxication. If untreated, death can result. Inhibited AChE can be reactivated by oximes, antidotes for OP exposure. However, OP intoxication caused by the nerve agent tabun (GA) is particularly resistant to oximes, which poorly reactivate GA-inhibited AChE. In an attempt to develop a rational strategy for the discovery and design of novel reactivators with lower toxicity and increased efficacy in reactivating GA-inhibited AChE, we developed the first in silico pharmacophore model for binding affinity of GA-inhibited AChE from a set of 11 oximes. Oximes were analyzed for stereoelectronic profiles and three-dimensional quantitative structure-activity relationship pharmacophores using ab initio quantum chemical and pharmacophore generation methods. Quantum chemical methods were sequentially used from semiempirical AM1 to hierarchical ab initio calculations to determine the stereoelectronic properties of nine oximes exhibiting affinity for binding to GA-inhibited AChE in vivo. The calculated stereoelectronic properties led us to develop the in silico pharmacophore model using CATALYST methodology. Specific stereoelectronic profiles including the distance between bisquarternary nitrogen atoms of the pyridinium ring in the oximes, hydrophilicity, surface area, nucleophilicity of the oxime oxygen, and location of the molecular orbitals on the isosurfaces have important roles for potencies for reactivating GA-inhibited AChE. The in silico pharmacophore model of oxime affinity for binding to GA-inhibited AChE was found to require a hydrogen bond acceptor, a hydrogen bond donor at the two terminal regions, and an aromatic ring in the central region of the oximes. The model was found to be well-correlated (R = 0.9) with experimental oxime affinity for binding to GA-inhibited AChE. Additional stereoelectronic features relating activity with

  8. Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis).

    PubMed Central

    Talesa, V; Grauso, M; Giovannini, E; Rosi, G; Toutant, J P

    1995-01-01

    Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M. Images Figure 2 Figure 3 Figure 4 PMID:7702560

  9. Syntheses of coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, Aβ aggregation, and β-secretase.

    PubMed

    Sun, Qi; Peng, Da-Yong; Yang, Sheng-Gang; Zhu, Xiao-Lei; Yang, Wen-Chao; Yang, Guang-Fu

    2014-09-01

    Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer's disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine-coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (Ki=16.7nM) against human AChE and about 2-fold lower potency (Ki=16.1nM) against BChE than tacrine (Ki=35.7nM for AChE, Ki=8.7nM for BChE), respectively. In addition, some of the tacrine-coumarin hybrids showed simultaneous inhibitory effects against both Aβ aggregation and β-secretase. We therefore conclude that tacrine-coumarin hybrid is an interesting multifunctional lead for the AD drug discovery.

  10. Downregulated expression of microRNA-124 in pediatric intestinal failure patients modulates macrophages activation by inhibiting STAT3 and AChE

    PubMed Central

    Xiao, Yong-Tao; Wang, Jun; Lu, Wei; Cao, Yi; Cai, Wei

    2016-01-01

    Intestinal inflammation plays a critical role in the pathogenesis of intestinal failure (IF). The macrophages are essential to maintain the intestinal homeostasis. However, the underlying mechanisms of intestinal macrophages activation remain poorly understood. Since microRNAs (miRNAs) have pivotal roles in regulation of immune responses, here we aimed to investigate the role of miR-124 in the activation of intestinal macrophages. In this study, we showed that the intestinal macrophages increased in pediatric IF patients and resulted in the induction of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The miRNA fluorescence in situ hybridization analysis showed that the expression of miR-124 significantly reduced in intestinal macrophages in IF patients. Overexpression of miR-124 was sufficient to inhibit intestinal macrophages activation by attenuating production of IL-6 and TNF-α. Further studies showed that miR-124 could directly target the 3′-untranslated region of both signal transducer and activator of transcription 3 (STAT3) and acetylcholinesterase (AChE) mRNAs, and suppress their protein expressions. The AChE potentially negates the cholinergic anti-inflammatory signal by hydrolyzing the acetylcholine. We here showed that intestinal macrophages increasingly expressed the AChE and STAT3 in IF patients when compared with controls. The inhibitors against to STAT3 and AChE significantly suppressed the lipopolysaccharides-induced IL-6 and TNF-α production in macrophages. Taken together, these findings highlight an important role for miR-124 in the regulation of intestinal macrophages activation, and suggest a potential application of miR-124 in pediatric IF treatment regarding as suppressing intestinal inflammation. PMID:27977009

  11. A microfluidic paper-based device to assess acetylcholinesterase activity.

    PubMed

    Liu, Chunye; Gomez, Frank A

    2017-04-01

    Neurotransmitters play key roles in cell-to-cell communication. These chemical messengers are involved in many functional processes, including growth, reproduction, memory, and behavior. In this communication, we describe a novel microfluidic paper-based analytical device (μPAD) to detect acetylcholinesterase (AChE) activity and inhibitor screening through a colorimetric analysis. The μPAD is easily fabricated via a wax printing process whereby wax is deposited onto the surface of chromatographic paper, and heated to create a hydrophobic barrier. Separate solutions of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and samples containing AChE and acetylthiocholine iodide (ATC) (or cysteine, Cys), respectively, are directly spotted onto the μPAD. DTNB and AChE/ATC (or Cys) flow towards each other where a reaction occurs to form the yellow colored 2-nitro-5-thiobenzoic acid anion (TNB(2-) ). The device is dried, scanned, and analyzed yielding a linear range of average inverse yellow intensities versus substrate concentration. An IC50 value (0.045 nM) with a known inhibitor, neostigmine bromide (NB), is obtained on the device. μPADs are low cost and easy to fabricate and have great potential to quantify neurotransmitter activity.

  12. Expression of acetylcholinesterase 1 is associated with brood rearing status in the honey bee, Apis mellifera.

    PubMed

    Kim, Young Ho; Kim, Ju Hyeon; Kim, Kyungmun; Lee, Si Hyeock

    2017-01-03

    Acetylcholinesterase 1 (AmAChE1) of the honey bee, Apis mellifera, has been suggested to have non-neuronal functions. A systematic expression profiling of AmAChE1 over a year-long cycle on a monthly basis revealed that AmAChE1 was predominantly expressed in both head and abdomen during the winter months and was moderately expressed during the rainy summer months. Interestingly, AmAChE1 expression was inhibited when bees were stimulated for brood rearing by placing overwintering beehives in strawberry greenhouses with a pollen diet, whereas it resumed when the beehives were moved back to the cold field, thereby suppressing brood rearing. In early spring, pollen diet supplementation accelerated the induction of brood-rearing activity and the inhibition of AmAChE1 expression. When active beehives were placed in a screen tent in late spring, thereby artificially suppressing brood-rearing activity, AmAChE1 was highly expressed. In contrast, AmAChE1 expression was inhibited when beehives were allowed to restore brood rearing by removing the screen, supporting the hypothesis that brood rearing status is a main factor in the regulation of AmAChE1 expression. Since brood rearing status is influenced by various stress factors, including temperature and diet shortage, our finding discreetly suggests that AmAChE1 is likely involved in the stress response or stress management.

  13. Expression of acetylcholinesterase 1 is associated with brood rearing status in the honey bee, Apis mellifera

    PubMed Central

    Kim, Young Ho; Kim, Ju Hyeon; Kim, Kyungmun; Lee, Si Hyeock

    2017-01-01

    Acetylcholinesterase 1 (AmAChE1) of the honey bee, Apis mellifera, has been suggested to have non-neuronal functions. A systematic expression profiling of AmAChE1 over a year-long cycle on a monthly basis revealed that AmAChE1 was predominantly expressed in both head and abdomen during the winter months and was moderately expressed during the rainy summer months. Interestingly, AmAChE1 expression was inhibited when bees were stimulated for brood rearing by placing overwintering beehives in strawberry greenhouses with a pollen diet, whereas it resumed when the beehives were moved back to the cold field, thereby suppressing brood rearing. In early spring, pollen diet supplementation accelerated the induction of brood-rearing activity and the inhibition of AmAChE1 expression. When active beehives were placed in a screen tent in late spring, thereby artificially suppressing brood-rearing activity, AmAChE1 was highly expressed. In contrast, AmAChE1 expression was inhibited when beehives were allowed to restore brood rearing by removing the screen, supporting the hypothesis that brood rearing status is a main factor in the regulation of AmAChE1 expression. Since brood rearing status is influenced by various stress factors, including temperature and diet shortage, our finding discreetly suggests that AmAChE1 is likely involved in the stress response or stress management. PMID:28045085

  14. Nanoparticle-based immunosensor with apoferritin templated metallic phosphate label for quantification of phosphorylated acetylcholinesterase

    SciTech Connect

    Du, Dan; Chen, Aiqiong; Xie, Yunying; Zhang, Aidong; Lin, Yuehe

    2011-05-15

    A new sandwich-like electrochemical immunosensor has been developed for quantification of organophosphorylated acetylcholinesterase (OP-AChE), an exposure biomarker of organophosphate pesticides and nerve agents. Zirconia nanoparticles (ZrO2 NPs) were anchored on a screen printed electrode (SPE) to preferably capture OP-AChE adducts by metal chelation with phospho-moieties, which was selectively recognized by lead phosphate-apoferritin labeled anti-AChE antibody (LPA-anti-AChE). The sandwich-like immunoreactions were performed among ZrO2 NPs, OP-AChE and LPA-anti-AChE to form ZrO2/OP-AChE/LPA-anti-AChE complex and the released lead ions were detected on a disposable SPE. The binding affinity was investigated by both square wave voltammetry (SWV) and quartz crystal microbalance (QCM) measurements. The proposed immunosensor yielded a linear response current over a broad OP-AChE concentrations range from 0.05 nM to 10 nM, with detection limit of 0.02 nM, which has enough sensitivity for monitoring of low-dose exposure to OPs. This method avoids the drawback of unavailability of commercial OP-specific antibody as well as amplifies detection signal by using apoferritin encoded metallic phosphate nanoparticle tags. This nanoparticle-based immunosensor offers a new method for rapid, sensitive, selective and inexpensive quantification of phosphorylated adducts for monitoring of OP pesticides and nerve agents exposures.

  15. Exploration of the Energy Landscape of Acetylcholinesterase by Molecular Dynamics Simulation.

    NASA Astrophysics Data System (ADS)

    McCammon, J. Andrew

    2002-03-01

    Proteins have rough energy landscapes. Often more states than just the ground state are occupied and have biological functions. It is essential to study these conformational substates and the dynamical transitions among them. Acetylcholinesterase (AChE) is an important enzyme that has biological functions including the termination of synaptic transmission signals. X-ray structures show that it has an active site that is accessible only via a long and narrow channel from its surface. Therefore the fact that acetylcholine and larger ligands can reach the active site is believed to reflect the protein's structural fluctuation. We carried out long molecular dynamics simulations to investigate the dynamics of AChE and its relation to biological function, and compared our results with experiments. The results reveal several "doors" that open intermittantly between the active site and the surface. Instead of having simple exponential decay correlation functions, the time series of these channels reveal complex, fractal gating between conformations. We also compared the AChE dynamics data with those from an AchE-fasciculin complex. (Fasciculin is a small protein that is a natural inhibitor of AChE.) The results show remarkable effects of the protein-protein interaction, including allosteric and dynamical inhibition by fasciculin besides direct steric blocking. More information and images can be found at http://mccammon.ucsd.edu

  16. Neuromuscular Junction Impairment in Amyotrophic Lateral Sclerosis: Reassessing the Role of Acetylcholinesterase

    PubMed Central

    Campanari, Maria-Letizia; García-Ayllón, María-Salud; Ciura, Sorana; Sáez-Valero, Javier; Kabashi, Edor

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a highly debilitating disease caused by progressive degeneration of motorneurons (MNs). Due to the wide variety of genes and mutations identified in ALS, a highly varied etiology could ultimately converge to produce similar clinical symptoms. A major hypothesis in ALS research is the “distal axonopathy” with pathological changes occurring at the neuromuscular junction (NMJ), at very early stages of the disease, prior to MNs degeneration and onset of clinical symptoms. The NMJ is a highly specialized cholinergic synapse, allowing signaling between muscle and nerve necessary for skeletal muscle function. This nerve-muscle contact is characterized by the clustering of the collagen-tailed form of acetylcholinesterase (ColQ-AChE), together with other components of the extracellular matrix (ECM) and specific key molecules in the NMJ formation. Interestingly, in addition to their cholinergic role AChE is thought to play several “non-classical” roles that do not require catalytic function, most prominent among these is the facilitation of neurite growth, NMJ formation and survival. In all this context, abnormalities of AChE content have been found in plasma of ALS patients, in which AChE changes may reflect the neuromuscular disruption. We review these findings and particularly the evidences of changes of AChE at neuromuscular synapse in the pre-symptomatic stages of ALS. PMID:28082868

  17. Coextracted dissolved organic carbon has a suppressive effect on the acetylcholinesterase inhibition assay.

    PubMed

    Neale, Peta A; Escher, Beate I

    2013-07-01

    The acetylcholinesterase (AChE) inhibition assay is frequently applied to detect organophosphates and carbamate pesticides in different water types, including dissolved organic carbon (DOC)-rich wastewater and surface water. The aim of the present study was to quantify the effect of coextracted DOC from different water samples on the commonly used enzyme-based AChE inhibition assay. Approximately 40% to 70% of DOC is typically recovered by solid-phase extraction, and this comprises not only organic micropollutants but also natural organic matter. The inhibition of the water extracts in the assay differed greatly from the expected mixture effects based on chemical analysis of organophosphates and carbamates. Binary mixture experiments with the known AChE inhibitor parathion and the water extracts showed reduced toxicity in comparison with predictions using the mixture models of concentration addition and independent action. In addition, the extracts and reference organic matter had a suppressive effect on a constant concentration of parathion. The present study thus indicated that concentrations of DOC as low as 2 mg carbon/L can impair the AChE inhibition assay and, consequently, that only samples with a final DOC concentration of less than 2 mgC /L are suitable for this assay. To check for potential suppression in environmental samples, standard addition experiments using an AChE-inhibiting reference compound are recommended.

  18. Mutations in the C-terminal domain of ColQ in endplate acetylcholinesterase deficiency compromise ColQ-MuSK interaction.

    PubMed

    Nakata, Tomohiko; Ito, Mikako; Azuma, Yoshiteru; Otsuka, Kenji; Noguchi, Yoichiro; Komaki, Hirofumi; Okumura, Akihisa; Shiraishi, Kazuhiro; Masuda, Akio; Natsume, Jun; Kojima, Seiji; Ohno, Kinji

    2013-07-01

    Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is mostly composed of an asymmetric form in which three tetramers of catalytic AChE subunits are linked to a triple helical collagen Q (ColQ). Mutations in COLQ cause endplate AChE deficiency. We report three patients with endplate AChE deficiency with five recessive COLQ mutations. Sedimentation profiles showed that p.Val322Asp and p.Arg227X, but not p.Cys444Tyr, p.Asp447His, or p.Arg452Cys, inhibit formation of triple helical ColQ. In vitro overlay of mutant ColQ-tailed AChE on muscle sections of Colq(-/-) mice revealed that p.Cys444Tyr, p.Asp447His, and p.Arg452Cys in the C-terminal domain (CTD) abrogate anchoring ColQ-tailed AChE to the NMJ. In vitro plate-binding assay similarly demonstrated that the three mutants inhibit binding of ColQ-tailed AChE to MuSK. We also confirmed the pathogenicity of p.Asp447His by treating Colq(-/-) mice with adeno-associated virus serotype 8 carrying mutant COLQ-p.Asp447His. The treated mice showed no improvement in motor functions and no anchoring of ColQ-tailed AChE at the NMJ. Electroporation of mutant COLQ harboring p.Cys444Tyr, p.Asp447His, and p.Arg452Cys into anterior tibial muscles of Colq(-/-) mice similarly failed to anchor ColQ-tailed AChE at the NMJ. We proved that the missense mutations in ColQ-CTD cause endplate AChE deficiency by compromising ColQ-MuSK interaction at the NMJ.

  19. From traditional European medicine to discovery of new drug candidates for the treatment of dementia and Alzheimer's disease: acetylcholinesterase inhibitors.

    PubMed

    Russo, P; Frustaci, A; Del Bufalo, A; Fini, M; Cesario, A

    2013-01-01

    The leading Alzheimer's disease (AD) therapeutics to date involves inhibitors of acetylcholinesterase (AChE), which should, in principle, elevate cholinergic signaling and limit inflammation. In spite of the effectiveness in 20%-30% of AD patients, more attention has been paid to find new anti-AChE agents from medicinal plants. Galanthamine, contained in the bulbs and flowers of Galanthus and related genera like Narcissus, represents a good example. The aim of this study is to review the role of possible AChE inhibitors (AChEI) present in plants traditionally used in European medicine for improving memory. Starting from Galanthamine, properties of Melissa species, Salvia officinalis, Arnica chamissonis and Ruta graveolens are discussed to point to the role of these plants as potential sources for the development of therapeutic agents for AD.

  20. In vivo and in vitro effects of fructose on rat brain acetylcholinesterase activity: an ontogenetic study.

    PubMed

    Guimarães, Carine A; Biella, Mairis S; Lopes, Abigail; Deroza, Pedro F; Oliveira, Mariana B; Macan, Tamires P; Streck, Emilio L; Ferreira, Gustavo C; Zugno, Alexandra I; Schuck, Patrícia F

    2014-12-01

    Increased fructose concentrations are the biochemical hallmark of fructosemia, a group of inherited disorders on the metabolic pathway of this sugar. The main clinical findings observed in patients affected by fructosemia include neurological abnormalities with developmental delay, whose pathophysiology is still undefined. In the present work we investigated the in vitro and in vivo effects of fructose on acetylcholinesterase (AchE) activity in brain structures of developing rats. For the in vitro experiments, fructose was added at increasing concentrations to the incubation medium. It was observed that fructose provoked an inhibition of acetylcholinesterase activity in cerebral cortex of 30-day-old-rats, even at low concentrations (0.1 mM). For the in vivo experiments, rats were killed 1 h after a single fructose administration (5 µmol/g). Control group received the same volume of saline solution. We found that AchE activity was increased in cerebral cortex of 30- and 60-day-old rats receiving fructose administration. Finally, we observed that AchE activity was unaffected by acute fructose administration in cerebral cortex, striatum or hippocampus of 15- and 90-day-old rats. The present data suggest that a disruption in cholinergic homeostasis may be involved in the pathophysiology of brain damage observed in young patients affected by fructosemia.

  1. Acetylcholinesterase inhibitory activity of lycopodane-type alkaloids from the Icelandic Lycopodium annotinum ssp. alpestre.

    PubMed

    Halldorsdottir, Elsa Steinunn; Jaroszewski, Jerzy W; Olafsdottir, Elin Soffia

    2010-02-01

    The aim of this study was to investigate structures and acetylcholinesterase inhibitory activities of lycopodane-type alkaloids isolated from an Icelandic collection of Lycopodium annotinum ssp. alpestre. Ten alkaloids were isolated, including annotinine, annotine, lycodoline, lycoposerramine M, anhydrolycodoline, gnidioidine, lycofoline, lannotinidine D, and acrifoline, as well as a previously unknown N-oxide of annotine. 1H and 13C NMR data of several of the alkaloids were provided for the first time. Solvent-dependent equilibrium constants between ketone and hemiketal form of acrifoline were determined. Conformation of acrifoline was characterized using NOESY spectroscopy and molecular modelling. The isolated alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Ligand docking studies based on mutated 3D structure of Torpedo californica acetylcholinesterase provided rationale for low inhibitory activity of the isolated alkaloids as compared to huperzine A or B, which are potent acetylcholinesterase inhibitors belonging to the lycodine class. Based on the modelling studies the lycopodane-type alkaloids seem to fit well into the active site gorge of the enzyme but the position of their functional groups is not compatible with establishing strong hydrogen bonding interactions with the amino acid residues that line the binding site. The docking studies indicate possibilities of additional functionalization of the lycopodane skeleton to render potentially more active analogues.

  2. Nerolidol-loaded nanospheres prevent behavioral impairment via ameliorating Na(+), K(+)-ATPase and AChE activities as well as reducing oxidative stress in the brain of Trypanosoma evansi-infected mice.

    PubMed

    Baldissera, Matheus D; Souza, Carine F; Grando, Thirssa H; Moreira, Karen L S; Schafer, Andressa S; Cossetin, Luciana F; da Silva, Ana P T; da Veiga, Marcelo L; da Rocha, Maria Izabel U M; Stefani, Lenita M; da Silva, Aleksandro S; Monteiro, Silvia G

    2017-02-01

    The aim of this study was to investigate the effect of nerolidol-loaded nanospheres (N-NS) on the treatment of memory impairment caused by Trypanosoma evansi in mice, as well as oxidative stress, and Na(+), K(+)-ATPase and acetylcholinesterase (AChE) activities in brain tissue. Animals were submitted to behavioral tasks (inhibitory avoidance task and open-field test) 4 days postinfection (PI). Reactive oxygen species (ROS) and thiobarbituric acid-reactive substance (TBARS) levels and catalase (CAT), superoxide dismutase (SOD), Na(+), K(+)-ATPase and AChE activities were measured on the fifth-day PI. T. evansi-infected mice showed memory deficit, increased ROS and TBARS levels and SOD and AChE activities, and decreased CAT and Na(+), K(+)-ATPase activities compared to uninfected mice. N-NS prevented memory impairment and oxidative stress parameters (except SOD activity), while free nerolidol (N-F) restored only CAT activity. Also, N-NS treatment was able to prevent alterations in Na(+), K(+)-ATPase and AChE activities caused by T. evansi infection. A significantly negative correlation was observed between memory and ROS production (p < 0.001; r = -0.941), as well as between memory and AChE activity (p < 0.05; r = -0.774). On the contrary, a significantly positive correlation between memory and Na(+), K(+)-ATPase activity was observed (p < 0.01; r = 0.844). In conclusion, N-NS was able to reverse memory impairment and to prevent increased ROS and TBARS levels due to amelioration of Na(+), K(+)-ATPase and AChE activities and to activation of the antioxidant enzymes, respectively. These results suggest that N-NS treatment may be a useful strategy to treat memory dysfunction and oxidative stress caused by T. evansi infection.

  3. A first principle study on the interaction between acetylcholinesterase and acetylcholine, and also rivastigmine in alzheimer's disease case

    NASA Astrophysics Data System (ADS)

    Khoirunisa, V.; Rusydi, F.; Kasai, H.; Gandaryus, A. G.; Dipojono, H. K.

    2016-08-01

    The catalytic activity of acetylcholinesterase enzyme (AChE) relates to the symptom progress in Alzheimer's disease. Interaction of AChE with rivastigmine (from the medicine) can reduce its catalytic activity toward acetylcholine to decelerate the progression of Alzheimer's disease. This research attempts to study the interaction between AChE and rivastigmine, and also acetylcholine (without the presence of rivastigmine) using density functional theory by simplifying the reaction occurs in the active site, which is assumed to be C2H5OH, C3N2H3(Ch3), and CH3COO-. The results suggest that AChE interacts easier with acetylcholine than with rivastigmine, which implies that the medicine does not effectively reduce the catalytic activity of AChE. At this stage, no experimental data is available to be compared with the calculation results. Nonetheless, this study has shown a good prospect to understand the AChE-substrate interaction using a first-principles calculation.

  4. Biosensor based on Butyrylcholinesterase for Detection of Carbofuran

    NASA Astrophysics Data System (ADS)

    Dey, Mousumi; Bhuvanagayathri, R.; Daniel, David K.

    2015-04-01

    Esterase enzymes play an important role in biology because they are responsible for the hydrolysis of choline esters. In their absence, the original state of the post synaptic membranes cannot be reestablished. Therefore, the aim of the work is to study the inhibiting action exerted by the group of compounds on these enzymes. Among these class of inhibiting compounds, pesticides are important because of the potential danger as a result of their large scale use in agriculture. Pesticides are generally determined using liquid or gas chromatography methods with various detection techniques. These methods are very sensitive and discriminating, however they require sample pretreatment such as extraction, preconcentration and clean up, which are skilled techniques and high cost treatment and also time consuming. In this study, acetyl cholinesterase and butyrylcholinesterase based biosensors have emerged as a promising tool for the detection and characterization of pesticides which are inhibitors of these enzymes. Although the physiological function of butyrylcholinesterase in comparison with acetyl cholinesterase is ambiguous, it has larger substrate specificity towards choline esters. Therefore, the development of a more selective electrode against choline, can lead to more sensitive determination of the inhibitor being investigated. Hence in the present work, a method based on inhibition of butyrylcholinesterase was attempted for quantification of carbofuran on the basis of cholinesterase inhibition. Butyrylcholinesterase with an activity of 10.2 units/mg was immobilized on a solid surface by cross linking with glutaraldehyde. The immobilized system was calibrated by correlating the inhibition of the butyrylcholinesterase activity with varying concentrations of the butyryl choline chloride and carbofuran. The sensing mechanism was investigated for its response to carbofuran concentrations ranging from 125 to 1,000 ppm. The effects of butyryl choline chloride

  5. Understanding the conformational flexibility and electrostatic properties of curcumin in the active site of rhAChE via molecular docking, molecular dynamics, and charge density analysis.

    PubMed

    Saravanan, Kandasamy; Kalaiarasi, Chinnasamy; Kumaradhas, Poomani

    2017-01-04

    Acetylcholinesterase (AChE) is an important enzyme responsible for Alzheimer's disease, as per report, keto-enol form of curcumin inhibits this enzyme. The present study aims to understand the binding mechanism of keto-enol curcumin with the recombinant human Acetylcholinesterase (rhAChE) from its conformational flexibility, intermolecular interactions, charge density distribution, and the electrostatic properties at the active site of rhAChE. To accomplish this, a molecular docking analysis of curcumin with the rhAChE was performed, which gives the structure and conformation of curcumin in the active site of rhAChE. Further, the charge density distribution and the electrostatic properties of curcumin molecule (lifted from the active site of rhAChE) were determined from the high level density functional theory (DFT) calculations coupled with the charge density analysis. On the other hand, the curcumin molecule was optimized (gas phase) using DFT method and further, the structure and charge density analysis were also carried out. On comparing the conformation, charge density distribution and the electrostatic potential of the active site form of curcumin with the corresponding gas phase form reveals that the above said properties are significantly altered when curcumin is present in the active site of rhAChE. The conformational stability and the interaction of curcumin in the active site are also studied using molecular dynamics simulation, which shows a large variation in the conformational geometry of curcumin as well as the intermolecular interactions.

  6. The butyrylcholinesterase knockout mouse a research tool in the study of drug sensitivity, bio-distribution, obesity and Alzheimer's disease.

    PubMed

    Duysen, Ellen G; Li, Bin; Lockridge, Oksana

    2009-05-01

    Butyrylcholinesterase (BChE) mutations common in the human population may result in complete or partial BChE deficiency, making the BChE knockout (KO) mouse a model for human deficiencies. The BChE KO mouse cannot tolerate standard doses of the muscle relaxant succinylcholine or the Alzheimer's disease drugs huperzine A and donepezil. It is resistant to the asthma drug bambuterol. The importance of BChE in detoxication of cocaine has been demonstrated by hepatotoxicity and cardiotoxicity in cocaine-challenged BChE KO mice. The BChE KO mouse becomes obese on a high-fat diet, suggesting a role for BChE in fat metabolism. BChE serves as a backup for acetylcholinesterase by hydrolyzing the neurotransmitter acetylcholine in acetylcholinesterase knockout mice. Imaging studies show that BChE injected intrathecally crosses the blood-brain barrier. Mice, but not humans, have carboxylesterase in their blood. Carboxylesterase obscures the role of BChE in detoxication of organophosphorus pesticides. Future studies will make a double knockout that has neither BChE nor carboxylesterase. The double knockout is expected to be unusually sensitive to the toxicity of organophosphorus pesticides. Knowledge of drug sensitivities in the mouse model of human BChE deficiency will aid in understanding adverse drug effects in humans.

  7. Generation of Recombinant Human AChE Op-Scavengers With Extended Circulatory Longevity

    DTIC Science & Technology

    2005-04-01

    AChE PEGylation results in a major reduction of the immunogenicity of the enzyme. In structure -function studies of AChE, we compared the reactivities...BChE). Extensive structural and biochemical analyses of over twenty forms of recombinant AChEs allowed us to determine an hierarchical pattern by...glycan structures that do not conform with the classical complex-type of oligosaccharides typical of animal cell proteins or which were entirely devoid of

  8. In vitro anti-acetylcholinesterase activity of an aqueous extract of Unicaria tomentosa and in silico study of its active constituents

    PubMed Central

    Chowdhury, Suman; Shivani; Kumar, Suresh

    2016-01-01

    Depletion of acetylcholine in the central nervous system (CNS) is responsible for memory loss and cognition deficit. Enzyme acetylcholinesterase (AChE) is responsible for destruction of acetylcholine (Ach) in the brain. Many herbal plant extracts have been investigated for their potential use in the treatment of Alzheimer’s disease (AD) by inhibiting AChE and upregulating the levels of Ach. The current study investigated the anti-acetylcholinesterase (AChE) activity of an aqueous extract of Unicaria tomentosa bark which has not been reported so far in the literature. The in vitro study of an aqueous extract of U. tomentosa showed maximum inhibition of 76.2±0.002 % at 0.4mg/ml of final concentration with an IC50 = 0.112 mg/ml. The mechanism of inhibition was elucidated by kinetic study which showed mixed type of inhibition, this might be due to the presence of various phytoconstituents such as oxindole alkaloids present in an aqueous extract. Based on molecular structure of phytoconstituents obtained from U. tomentosa known from the relevant literature, in-silico molecular docking study was performed against AChE protein to validate the results. PMID:28149044

  9. Combining in silico and in vitro approaches to evaluate the acetylcholinesterase inhibitory profile of some commercially available flavonoids in the management of Alzheimer's disease.

    PubMed

    Kuppusamy, Asokkumar; Arumugam, Madeswaran; George, Sonia

    2017-02-01

    The current objective of the study is to identify inhibitory affinity potential of the certain commercially available flavonoids, against crystal structure of acetylcholinesterase (AChE) enzyme using in silico and in vitro studies. The inhibitory profiles of the compounds have been compared with standard AChE inhibitor donepezil. In the docking studies, conformational site analysis and docking parameters like binding energy, inhibition constant and intermolecular energy were determined using AutoDock 4.2. Docking studies conducted with diosmin, silibinin, scopoletin, taxifolin and tricetin exhibited tight binding forces prevailing with the enzyme than between donepezil. Based on the in silico studies, compounds were selected for the in vitro AChE inhibitory assay. In vitro results showed that all the selected flavonoids displayed excellent concentration-dependant inhibition of AChE. Scopoletin was found to be the most potent and specific inhibitor of the enzyme with IC50 values of 10.18±0.68μM. Scopoletin showed several strong hydrogen bonds to several important amino acid residues against target enzyme. A number of hydrophobic interactions could also explain the potency of the compounds to inhibit AChE. These molecular docking and in vitro analyses could lead to the further development of potent acetylcholinesterase inhibitors for the treatment of Alzheimer's disease.

  10. In vitro anti-acetylcholinesterase activity of an aqueous extract of Unicaria tomentosa and in silico study of its active constituents.

    PubMed

    Chowdhury, Suman; Shivani; Kumar, Suresh

    2016-01-01

    Depletion of acetylcholine in the central nervous system (CNS) is responsible for memory loss and cognition deficit. Enzyme acetylcholinesterase (AChE) is responsible for destruction of acetylcholine (Ach) in the brain. Many herbal plant extracts have been investigated for their potential use in the treatment of Alzheimer's disease (AD) by inhibiting AChE and upregulating the levels of Ach. The current study investigated the anti-acetylcholinesterase (AChE) activity of an aqueous extract of Unicaria tomentosa bark which has not been reported so far in the literature. The in vitro study of an aqueous extract of U. tomentosa showed maximum inhibition of 76.2±0.002 % at 0.4mg/ml of final concentration with an IC50 = 0.112 mg/ml. The mechanism of inhibition was elucidated by kinetic study which showed mixed type of inhibition, this might be due to the presence of various phytoconstituents such as oxindole alkaloids present in an aqueous extract. Based on molecular structure of phytoconstituents obtained from U. tomentosa known from the relevant literature, in-silico molecular docking study was performed against AChE protein to validate the results.

  11. Acetylcholinesterase as a Biomarker in Environmental and Occupational Medicine: New Insights and Future Perspectives

    PubMed Central

    Caricato, Roberto; Calisi, Antonio; Giordano, Maria Elena; Schettino, Trifone

    2013-01-01

    Acetylcholinesterase (AChE) is a key enzyme in the nervous system. It terminates nerve impulses by catalysing the hydrolysis of neurotransmitter acetylcholine. As a specific molecular target of organophosphate and carbamate pesticides, acetylcholinesterase activity and its inhibition has been early recognized to be a human biological marker of pesticide poisoning. Measurement of AChE inhibition has been increasingly used in the last two decades as a biomarker of effect on nervous system following exposure to organophosphate and carbamate pesticides in occupational and environmental medicine. The success of this biomarker arises from the fact that it meets a number of characteristics necessary for the successful application of a biological response as biomarker in human biomonitoring: the response is easy to measure, it shows a dose-dependent behavior to pollutant exposure, it is sensitive, and it exhibits a link to health adverse effects. The aim of this work is to review and discuss the recent findings about acetylcholinesterase, including its sensitivity to other pollutants and the expression of different splice variants. These insights open new perspective for the future use of this biomarker in environmental and occupational human health monitoring. PMID:23936791

  12. Acetylcholinesterase as a biomarker in environmental and occupational medicine: new insights and future perspectives.

    PubMed

    Lionetto, Maria Giulia; Caricato, Roberto; Calisi, Antonio; Giordano, Maria Elena; Schettino, Trifone

    2013-01-01

    Acetylcholinesterase (AChE) is a key enzyme in the nervous system. It terminates nerve impulses by catalysing the hydrolysis of neurotransmitter acetylcholine. As a specific molecular target of organophosphate and carbamate pesticides, acetylcholinesterase activity and its inhibition has been early recognized to be a human biological marker of pesticide poisoning. Measurement of AChE inhibition has been increasingly used in the last two decades as a biomarker of effect on nervous system following exposure to organophosphate and carbamate pesticides in occupational and environmental medicine. The success of this biomarker arises from the fact that it meets a number of characteristics necessary for the successful application of a biological response as biomarker in human biomonitoring: the response is easy to measure, it shows a dose-dependent behavior to pollutant exposure, it is sensitive, and it exhibits a link to health adverse effects. The aim of this work is to review and discuss the recent findings about acetylcholinesterase, including its sensitivity to other pollutants and the expression of different splice variants. These insights open new perspective for the future use of this biomarker in environmental and occupational human health monitoring.

  13. Monooxime reactivators of acetylcholinesterase with (E)-but-2-ene linker: preparation and reactivation of tabun- and paraoxon-inhibited acetylcholinesterase.

    PubMed

    Musilek, Kamil; Holas, Ondrej; Jun, Daniel; Dohnal, Vlastimil; Gunn-Moore, Frank; Opletalova, Veronika; Dolezal, Martin; Kuca, Kamil

    2007-11-01

    Acetylcholinesterase reactivators are crucial antidotes for the treatment of organophosphate intoxication. Fifteen new monooxime reactivators of acetylcholinesterase with a (E)-but-2-ene linker were developed in an effort to extend the properties of K-oxime (E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide (K203). The known reactivators (pralidoxime, HI-6, obidoxime, K075, K203) and the new compounds were tested in vitro on a model of tabun- and paraoxon-inhibited AChE. Monooxime reactivators were not able to exceed the best known compounds for tabun poisoning, but some of them did show reactivation comparable with known compounds for paraoxon poisoning. However, extensive differences were found by a SAR study for various substitutions on the non-oxime part of the reactivator molecule.

  14. Evaluation of monoquaternary pyridinium oximes potency to reactivate tabun-inhibited human acetylcholinesterase.

    PubMed

    Odzak, Renata; Calić, Maja; Hrenar, Tomica; Primozic, Ines; Kovarik, Zrinka

    2007-04-20

    Monoquaternary N-benzyl-4-hydroxyiminomethylpyridinium bromide (Py-4-H) and its analogous with diverse substituents introduced into the phenyl ring (Py-4-CH(3), Py-4-Br, Py-4-Cl and Py-4-NO(2)) were synthesized in order to examine their potency as reactivators of tabun-inhibited human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7). Within 24h, the reactivation of tabun-inhibited AChE reached 80% with Py-4-CH(3), Py-4-Br and Py-4-Cl, 40% with Py-4-NO(2), and 30% with Py-4-H. The overall reactivation rate constants were up to 5.0min(-1)M(-1). All oximes inhibited human AChE reversibly, and the inhibition potency increased in the following order Py-4-BrAChE. Docking studies were carried out to elucidate the differences in oximes potency. The orientations of all studied oximes in the active site of human AChE have been proposed by flexible ligand docking with AutoDock 3.0. Analyses of the obtained complexes revealed the presence of numerous hydrogen bonds and close contacts between the oximes and the residues in the active site. Final docked energies predicted correctly the relative order of the inhibition potency of compounds (except in the case of Py-4-CH(3)) as well as the most probable orientation of the best reactivator, Py-4-Br, which can result in an attack on the phosphorus atom of the tabun-phosphorylated human AChE.

  15. Russian VX: inhibition and reactivation of acetylcholinesterase compared with VX agent.

    PubMed

    Kuca, Kamil; Jun, Daniel; Cabal, Jiri; Hrabinova, Martina; Bartosova, Lucie; Opletalova, Veronika

    2006-04-01

    Organophosphorus compounds such as nerve agents inhibit, practically irreversibly, cholinesterases by their phosphorylation in the active site of these enzymes. Current antidotal treatment used in the case of acute nerve agent intoxications consists of combined administration of anticholinergic drug (usually atropine) and acetylcholinesterase (AChE, EC 3.1.1.7) reactivator (HI-6, obidoxime, pralidoxime), which from a chemical view is a derivative from the group of pyridinium or bispyridinium aldoximes (commonly called "oxime"). Oximes counteract acetylcholine increase, resulting from AChE inhibition. In the human body environment these compounds are powerful nucleophiles and are able to break down the bond between AChE and nerve agent molecule. This process leads to renewal of enzyme functionality -- to its reactivation. The usefulness of oxime in the reactivation process depends on its chemical structure and on the nerve agent whereby AChE is inhibited. Due to this fact, selection of suitable reactivator in the treatment of intoxications is very important. In our work, we have compared differences in the in vitro inhibition potency of VX and Russian VX on rat, pig and human brain, and subsequently we have tested reactivation of rat brain cholinesterase inhibited by these agents using oxime HI-6, obidoxime, pralidoxime, trimedoxime and methoxime. The results showed that no major differences in the reactivation process of both VX and Russian VX-inhibited cholinesterase. The similarity in reactivation was caused by analogous chemical structure of either nerve agent; and that oxime HI-6 seems to be the most effective reactivator tested, which confirms that HI-6 is currently the most potent reactivator of AChE inhibited by nerve agents. The results obtained in our study should be considered in the future development of new AChE reactivators.

  16. Chromatographic preparation and kinetic analysis of interactions between tabun enantiomers and acetylcholinesterase.

    PubMed

    Tenberken, O; Thiermann, H; Worek, F; Reiter, G

    2010-06-02

    The easy accessibility to highly toxic OP (organophosphorus)-type chemical warfare agents (nerve agents) underlines the necessity for an effective medical treatment. Acute OP toxicity is primarily caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment. However, the commercially available compounds, obidoxime and pralidoxime, are considered rather ineffective against various nerve agents, including tabun. OP-type chemical warfare agents include an asymmetrical P-atom and consist of at least two stereoisomers. Previous studies with the nerve agents sarin and soman showed marked differences between (-)- and (+)-P isomers regarding AChE inhibition and stability in biological matrices. Hence, stereoselectivity is a key parameter for the development of optimized treatment. In the present study, the tabun enantiomers were isolated by semi-preparative liquid-chromatography (LC) with offline analysis by GC-PCI-MS and final characterization of optical purity (99.98% (-)-tabun and 99.83% (+)-tabun) and specific optical rotation. The inhibition and reactivation kinetics of the tabun enantiomers were determined with human and swine AChE and the aging kinetics with human AChE. The results show a large difference in the inhibitory potency between (-)- and (+)-tabun. The determination of reactivation and aging kinetics indicates that both reactions are at least in part determined by the residual (-)-tabun contamination (0.17%) of the (+)-tabun preparation. These data provide further insight into the kinetic interactions between tabun enantiomers and AChE and may contribute to the development of more effective treatment options.

  17. Exposure to Acetylcholinesterase Inhibitors Alters the Physiology and Motor Function of Honeybees

    PubMed Central

    Williamson, Sally M.; Moffat, Christopher; Gomersall, Martha A. E.; Saranzewa, Nastja; Connolly, Christopher N.; Wright, Geraldine A.

    2013-01-01

    Cholinergic signaling is fundamental to neuromuscular function in most organisms. Sub-lethal doses of neurotoxic pesticides that target cholinergic signaling can alter the behavior of insects in subtle ways; their influence on non-target organisms may not be readily apparent in simple mortality studies. Beneficial arthropods such as honeybees perform sophisticated behavioral sequences during foraging that, if influenced by pesticides, could impair foraging success and reduce colony health. Here, we investigate the behavioral effects on honeybees of exposure to a selection of pesticides that target cholinergic signaling by inhibiting acetylcholinesterase (AChE). To examine how continued exposure to AChE inhibitors affected motor function, we fed adult foraging worker honeybees sub-lethal concentrations of these compounds in sucrose solution for 24 h. Using an assay for locomotion in bees, we scored walking, stopped, grooming, and upside down behavior continuously for 15 min. At a 10 nM concentration, all the AChE inhibitors caused similar effects on behavior, notably increased grooming activity and changes in the frequency of bouts of behavior such as head grooming. Coumaphos caused dose-dependent effects on locomotion as well as grooming behavior, and a 1 μM concentration of coumaphos induced symptoms of malaise such as abdomen grooming and defecation. Biochemical assays confirmed that the four compounds we assayed (coumaphos, aldicarb, chlorpyrifos, and donepezil) or their metabolites acted as AChE inhibitors in bees. Furthermore, we show that transcript expression levels of two honeybee AChE inhibitors were selectively upregulated in the brain and in gut tissues in response to AChE inhibitor exposure. The results of our study imply that the effects of pesticides that rely on this mode of action have subtle yet profound effects on physiological effects on behavior that could lead to reduced survival. PMID:23386834

  18. Pre-treatment with ebselen and vitamin E modulate acetylcholinesterase activity: interaction with demyelinating agents.

    PubMed

    Mazzanti, Cinthia M; Spanevello, Roselia; Ahmed, Musthaq; Pereira, Luciane B; Gonçalves, Jamile F; Corrêa, Maisa; Schmatz, Roberta; Stefanello, Naiara; Leal, Daniela B R; Mazzanti, Alexandre; Ramos, Adriano T; Martins, Tessie B; Danesi, Cristiane Cademartori; Graça, Dominguita L; Morsch, Vera M; Schetinger, Maria Rosa C

    2009-02-01

    The ethidium bromide (EB) demyelinating model was associated with vitamin E (Vit E) and ebselen (Ebs) treatment to evaluate acetylcholinesterase (AChE) activity in the striatum (ST), hippocampus (HP), cerebral cortex (CC) and erythrocytes. Rats were divided into seven groups: I-Control (saline), II-(canola); III-(Ebs), IV-(Vit E); V-(EB); VI-(EB+Ebs) and VII-(EB+Vit E). At 3 days after the EB injection, AChE activity in the CC and HC was significantly reduced in groups III, IV, V, VI and VII (p<0.05) and in the ST it was reduced in groups III and V (p<0.05) when compared to the control group. At 21 days after the EB injection, AChE activity in the CC was significantly reduced in groups III, IV and V, while in groups VI and VII a significant increase was observed when compared to the control group. In the HC and ST, AChE activity was significantly reduced in groups V, VI and VII when compared to the control group (p<0.05). In the erythrocytes, at 3 days after the EB injection, AChE activity was significantly reduced in groups III, IV, V, VI and VII and at 21 days there was a significant reduction only in groups VI and VII (p<0.05) when compared to the control group. In conclusion, this study demonstrated that Ebs and Vit E interfere with the cholinergic neurotransmission by altering AChE activity in the different brain regions and in the erythrocytes. Furthermore, treatment with Vit E and Ebs protected against the demyelination lesion caused by EB. In this context, we can suggest that ebselen and Vit E should be considered potential therapeutics and scientific tools to be investigated in brain disorders associated with demyelinating events.

  19. Evaluation of a Brain Acetylcholinesterase Extraction Method and Kinetic Constants after Methyl-Paraoxon Inhibition in Three Brazilian Fish Species

    PubMed Central

    Freitas, A. P.; Santos, C. R.; Sarcinelli, P. N.; Hauser-Davis, R. A.; Lopes, R. M.

    2016-01-01

    Acetylcholinesterase (AChE) is an important enzyme in the control of the neuronal action potential and sensitive to organophosphate inhibition. Brain fish AChE is less sensitive to organophosphate inhibition than AChE from terrestrial animals, although this sensitivity is variable among species and has not yet been fully evaluated in fish species. In this setting, inhibition kinetic constants for progressive irreversible inhibition of brain acetylcholinesterase due to methyl-paraoxon exposure were determined in three fish species (Mugil liza, Genidens genidens and Lagocephalus laevigatus) and hen (Gallus domesticus). Enzyme extraction using a detergent was shown to be adequate, and samples presented activity inhibition in high substrate concentrations and suppression of inhibition by methyl-paraoxon in the presence of the substrate, similar to kinetic patterns from purified enzyme preparations. Catfish (G. genidens) AChE presented the highest sensitivity among the evaluated fish species (IC50 = 1031.20 nM ± 63.17) in comparison to M. liza and L. laevigatus (IC50: 2878.83 ± 421.94 and 2842.5 ± 144.63 nM respectively). The lower dissociation constant (Kd = 20.3 ± 2.95 μM) of catfish AChE showed greater enzyme affinity for methyl-paraoxon, explaining this species higher sensitivity to organophosphates. Hen AChE presented higher ki (900.57 ± 65.3 mM-1min-1) and, consequently, greater sensitivity to methyl-paraoxon, explained by a lower Kd (0.6 ± 0.13 μM). Furthermore, hen AChE did not differentiate between the propionylthiocholine and acetylthiocholine substrates, indicating easier access of methyl-paraoxon to the hen enzyme activity site. The results obtained herein indicate a suitable extraction of AChE and, despite different inhibition kinetic constants, demonstrate that fish AChE is less sensitive to methyl-paraoxon, probably due to reduced access to the catalytic center which provides greater enzyme substrate selectivity. PMID:27655611

  20. Inhibitor profile of bis(n)-tacrines and N-methylcarbamates on acetylcholinesterase from Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi

    PubMed Central

    Swale, Daniel R.; Tong, Fan; Temeyer, Kevin B.; Li, Andrew; Lam, Polo C-H.; Totrov, Maxim M.; Carlier, Paul R.; Pérez de León, Adalberto A.; Bloomquist, Jeffrey R.

    2013-01-01

    The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAChE) compared to human and bovine AChE, in order to identify divergent pharmacology that might lead to selective inhibitors. Results indicate that BmAChE has low sensitivity (IC50 = 200 μM) toward tacrine, a monovalent catalytic site inhibitor with sub micromolar blocking potency in all previous species tested. Similarly, a series of bis(n)-tacrine dimer series, bivalent inhibitors and peripheral site AChE inhibitors possess poor potency toward BmAChE. Molecular homology models suggest the rBmAChE enzyme possesses a W384F orthologous substitution near the catalytic site, where the larger tryptophan side chain obstructs the access of larger ligands to the active site, but functional analysis of this mutation suggests it only partially explains the low sensitivity to tacrine. In addition, BmAChE1 and PpAChE have low nanomolar sensitivity to some experimental carbamate anticholinesterases originally designed for control of the malaria mosquito, Anopheles gambiae. One experimental compound, 2-((2-ethylbutyl)thio)phenyl methylcarbamate, possesses >300-fold selectivity for BmAChE1 and PpAChE over human AChE, and a mouse oral LD50 of >1500 mg/kg, thus providing an excellent new lead for vector control. PMID:24187393

  1. Effect of pesticide exposure on acetylcholinesterase activity in subsistence farmers from Campeche, Mexico.

    PubMed

    Rendón von Osten, Jaime; Epomex, Centro; Tinoco-Ojanguren, Rolando; Soares, Amadeu M V M; Guilhermino, Lucia

    2004-08-01

    The authors surveyed agricultural production methods and pesticide use among subsistence farmers (campesinos) in 4 rural communities of Campeche, Mexico. Self-reports of symptoms of poisoning resulting from occupational pesticide exposure were elicited by questionnaire (N = 121), and acetylcholinesterase (AChE) activity during insecticide use was evaluated from blood samples (N = 127). In individuals from 2 of the 4 communities, AChE activity was significantly lower (p < 0.05) than the mean of activity determined for individuals in a reference group. Results of this study show that erythrocyte AChE inhibition provides a good biomarker of exposure to organophosphate pesticides in field studies with human populations. Carbamates, particularly carbofuran, seem to be more associated with exuberant and diversified symptomatology of pesticide exposure than organophosphates. Studies in field communities where both carbamates and organophosphates are suspected to exist should include blood AChE determinations, symptomatology surveys, and socioeconomic questionnaires. The authors recommend that the Mexican National Health Ministry authorities specify additional provisions regarding the use of protective equipment and the adoption of other safety practices during field work, increase information campaigns about the risks of pesticide use and the value of safety practices, and increase programs of medical monitoring and assistance for rural communities dealing with pesticides.

  2. Influence of water temperature on acetylcholinesterase activity in the pacific tree frog (Hyla regilla)

    USGS Publications Warehouse

    Johnson, Catherine S.; Schwarzbach, Steven E.; Henderson, John D.; Wilson, Barry W.; Tjeerdema, Ronald S.

    2005-01-01

    This investigation evaluated whether acetylcholinesterase (AChE) in Pacific tree frogs (Hyla regilla) from different geographical locations was influenced by different temperatures during early aquatic life stages, independent of pesticide exposure. Tadpoles were collected from both a California coastal pond and a Sierra Nevada mountain range pond, USA. Groups of frogs from each location were raised in temperatures representative of either the Sierra Nevada (8°C) or the coastal (19°C) location. Metamorphs from both locations raised as tadpoles at 19°C had AChE activities of 42.3 and 38.7 nm/min/mg protein, while those raised as tadpoles at 8°C had activities of 26.9 and 28.2 nm/min/mg protein. A two-way analysis of variance revealed temperature to be the significant factor in determining AChE activity (F = 22.3, p < 0.001), although origin was not important (F = 0.09, p = 0.75). Interpretations regarding the influence of pesticides upon AChE activity in Pacific tree frogs must consider the influence of environmental temperature to enable cross-population comparisons.

  3. Triterpenoids with acetylcholinesterase inhibition from Chuquiraga erinacea D. Don. subsp. erinacea (Asteraceae).

    PubMed

    Gurovic, María Soledad; Castro, María Julia; Richmond, Victoria; Faraoni, María Belén; Maier, Marta S; Murray, Ana Paula

    2010-04-01

    A bioactivity-guided approach was taken to identify the acetylcholinesterase (AChE) inhibitory agents in the ethanolic extract of Chuquiraga erinacea D. Don. subsp. erinacea leaves using a bioautographic method. This permitted the isolation of the pentacyclic triterpenes calenduladiol (1), faradiol (2), heliantriol B2 (3), lupeol (4), and a mixture of alpha-and beta-amyrin ( 5A and 5B) as active constituents. Pseudotaraxasterol (6) and taraxasterol (7) were also isolated from this extract and showed no activity at the same analytical conditions. Compound 1 showed the highest AChE inhibitory activity with 31.2 % of inhibition at 0.5 mM. Looking forward to improve the water solubility of the active compounds, the sodium sulfate ester of 1 was prepared by reaction with the (CH3)3N.SO3 complex. The semisynthetic derivative disodium calenduladiol disulfate (8) elicited higher AChE inhibition than 1 with 94.1 % of inhibition at 0.5 mM (IC (50) = 0.190 +/- 0.003 mM). Compounds 1, 2, 3, 5, 6, and 7 are reported here for the first time in C. erinacea. This is the first report of AChE inhibition from calenduladiol (1) as well as from a sulfate derived from a natural product.

  4. Acetylcholinesterase and metallothionein in oysters (Crassostrea corteziensis) from a subtropical Mexican Pacific estuary.

    PubMed

    Bernal-Hernández, Y Y; Medina-Díaz, I M; Robledo-Marenco, M L; Velázquez-Fernández, J B; Girón-Pérez, M I; Ortega-Cervantes, L; Maldonado-Vázquez, W A; Rojas-García, A E

    2010-04-01

    Substantial efforts have been devoted to developing and applying biomarkers for ecological risk assessment. Bivalve mollusks, such as mussels and oysters, are commonly used in environmental monitoring programs because of their wide geographical distribution, great sensitivity to environmental pollutants, and ability to accumulate anthropogenically derived chemicals at a high rate. Acetylcholinesterase (AChE) activity and metallothionein (MT's) content are representative specific biomarkers that indicate the presence of anticholinesterasic compounds (like organophosphorus and carbamate pesticides) and metals, respectively. The aim of this study was to evaluate AChE activity and MT's content in Crassostrea corteziensis from Boca de Camichín estuary. The results obtained here showed that AChE activity was 65% lower in oysters from Boca de Camichín than in control organisms. In contrast, MT's content in collected organisms was not statistically different from that in control organisms. AChE activity and MT's content in oysters could be used as early biomarkers of effects and exposure to pesticides and heavy metals, respectively, in aquatic environments.

  5. Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques.

    PubMed

    Gupta, Shikhar; Fallarero, Adyary; Järvinen, Päivi; Karlsson, Daniela; Johnson, Mark S; Vuorela, Pia M; Mohan, C Gopi

    2011-02-15

    Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer's disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the β-amyloid (Aβ) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads--Specs1 (IC(50)=3.279 μM) and Spec2 (IC(50)=5.986 μM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity.

  6. β-glucan attenuated scopolamine induced cognitive impairment via hippocampal acetylcholinesterase inhibition in rats.

    PubMed

    Haider, Ali; Inam, Wali; Khan, Shahab Ali; Hifza; Mahmood, Wajahat; Abbas, Ghulam

    2016-08-01

    β-glucan (polysaccharide) rich diet has been reported to enhance cognition in humans but the mechanism remained elusive. Keeping this in mind, the present study was designed to investigate the interaction of β-glucan with central cholinergic system. Briefly, in-silico analysis revealed promising interactions of β-glucan with the catalytic residues of acetylcholinesterase (AChE) enzyme. In line with this outcome, the in vitro assay (Ellman's method) also exhibited inhibition of AChE by β-glucan (IC50=0.68±0.08μg/µl). Furthermore, the in vivo study (Morris water maze) showed significant dose dependent reversal of the amnesic effect of scopolamine (2mg/kg i.p.) by β-glucan treatment (5, 25, 50 and 100mg/kg, i.p.). Finally, the hippocampi of aforementioned treated animals also revealed dose dependent inhibition of AChE enzyme. Hence, it can be deduced that β-glucan possesses potential to enhance central cholinergic tone via inhibiting AChE enzyme. In conclusion, the present study provides mechanistic insight to the cognition enhancing potential of β-glucan. Keeping in mind its dietary use and abundance in nature, it can be considered as economic therapeutic option against cognitive ailments associated with decline in cholinergic neurotransmission.

  7. Chemical composition, aroma evaluation, and inhibitory activity towards acetylcholinesterase of essential oils from Gynura bicolor DC.

    PubMed

    Miyazawa, Mitsuo; Nakahashi, Hiroshi; Usami, Atsushi; Matsuda, Naoki

    2016-04-01

    The compositions of the essential oils obtained from leaves and stems of Gynura bicolor DC. were analyzed by GC-MS. One hundred eight components of these oils were identified. (E)-β-caryophyllene (31.42 %), α-pinene (17.11 %), and bicyclogermacrene (8.09 %) were found to be the main components of the leaf oil, while α-pinene (61.42 %), β-pinene (14.39 %), and myrcene (5.10 %) were the major constituents of the stem oil. We found 73 previously unidentified components in these oils from G. bicolor. The oils were also subjected to odor evaluation. Eleven and 12 aroma-active compounds were detected in the leaf and stem oils, respectively. The abilities of these oils to inhibit acetylcholinesterase (AChE) activity were determined. The sesquiterpenoids in the oils were found to inhibit AChE activity more strongly than the monoterpenoids in the oils did. It was suggested that the three main components in each essential oil act synergistically against AChE activity. These results show that the essential oils obtained from G. bicolor are a good dietary source of AChE activity inhibition.

  8. Acetylcholinesterase inhibitory activity of Thai traditional nootropic remedy and its herbal ingredients.

    PubMed

    Tappayuthpijarn, Pimolvan; Itharat, Arunporn; Makchuchit, Sunita

    2011-12-01

    The incidence of Alzheimer disease (AD) is increasing every year in accordance with the increasing of elderly population and could pose significant health problems in the future. The use of medicinal plants as an alternative prevention or even for a possible treatment of the AD is, therefore, becoming an interesting research issue. Acetylcholinesterase (AChE) inhibitors are well-known drugs commonly used in the treatment of AD. The aim of the present study was to screen for AChE inhibitory activity of the Thai traditional nootropic recipe and its herbal ingredients. The results showed that ethanolic extracts of four out of twenty-five herbs i.e. Stephania pierrei Diels. Kaempfera parviflora Wall. ex Baker, Stephania venosa (Blume) Spreng, Piper nigrum L at 0.1 mg/mL showed % AChE inhibition of 89, 64, 59, 50; the IC50 were 6, 21, 29, 30 microg/mL respectively. The other herbs as well as combination of the whole recipe had no synergistic inhibitory effect on AChE activity. However some plants revealed antioxidant activity. More research should have be performed on this local wisdom remedy to verify the uses in scientific term.

  9. Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A from Sea Urchin Scaphechinus mirabilis

    PubMed Central

    Lee, Sung Ryul; Pronto, Julius Ryan D.; Sarankhuu, Bolor-Erdene; Ko, Kyung Soo; Rhee, Byoung Doo; Kim, Nari; Mishchenko, Natalia P.; Fedoreyev, Sergey A.; Stonik, Valentin A.; Han, Jin

    2014-01-01

    Echinochrome A (EchA) is a dark-red pigment of the polyhydroxynaphthoquinone class isolated from sea urchin Scaphechinus mirabilis. Acetylcholinesterase (AChE) inhibitors are used in the treatment of various neuromuscular disorders, and are considered as strong therapeutic agents for the treatment of Alzheimer’s disease (AD). Although EchA is clinically used to treat ophthalmic diseases and limit infarct formation during ischemia/reperfusion injury, anti-AChE effect of EchA is still unknown. In this study, we investigated the anti-AChE effect of EchA in vitro. EchA and its exhausted form which lost anti-oxidant capacity did not show any significant cytotoxicy on the H9c2 and A7r5 cells. EchA inhibited AChE with an irreversible and uncompetitive mode. In addition, EchA showed reactive oxygen species scavenging activity, particularly with nitric oxide. These findings indicate new therapeutic potential for EchA in treating reduced acetylcholine-related diseases including AD and provide an insight into developing new AChE inhibitors. PMID:24918454

  10. Acrylonitrile has Distinct Hormetic Effects on Acetyl-Cholinesterase Activity in Mouse Brain and Blood that are Modulated by Ethanol

    PubMed Central

    Yuanqing, He; Suhua, Wang; Guangwei, Xing; Chunlan, Ren; Hai, Qian; Wenrong, Xu; Rongzhu, Lu; Aschner, Michael; Milatovic, Dejan

    2013-01-01

    Acrylonitrile(AN) is a neurotoxin both in animals and humans, but its effects on acetylcholinesterase (AChE) activity remain controversial. This study aimed to determine the dose-response effects of AN on AChE activity and the modulatory role of ethanol pre-treatment. A total of 144 Kunming mice were randomly divided into 18 groups: nine groups received 5% ethanol in their drinking water, and the remaining nine groups received regular tap water. One week later, both the ethanol and tap water only groups were given an intraperitoneal injection of AN at the following doses: 0 (control), 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 or 20 mg AN/kg body weight. AChE activity was determined on whole blood and brain 24 h later. Blood AChE activity was higher in AN-injected mice than in controls at all doses. AChE activity in blood increased in a dose-dependent manner, peaking at 0.156 mg/kg, after which a gradual decrease ensued, displaying a β-typed dose-response relationship. In contrast, brain AChE activity, following a single AN injection, was consistently lower than in control mice, and continued to fall up to a dose of 0.313 mg/kg, and thereafter increased gradually with higher doses. Mice receiving a 20 mg/kg dose of AN exhibited AChE brain activity indistinguishable from that of control mice, demonstrating a typical U-typed dose-response relationship. The activity of AChE in the blood and brain of the AN + ethanol-treated groups displayed a shift to the right, and the magnitude of the decrease in AChE activity induced by AN was attenuated relative to the AN-only group. These results suggest that AN affects AChE activity in both mouse blood and brain in a hormetic manner. Pretreatment with ethanol modifies the effect of AN on AChE, indicating that parent AN has a more prominent role than its metabolites in modulating enzyme activity. PMID:23550232

  11. Acetylcholinesterase-reduced graphene oxide hybrid films for organophosphorus neurotoxin sensing via quartz crystal microbalance

    NASA Astrophysics Data System (ADS)

    Tang, Shi; Ma, Wenying; Xie, Guangzhong; Su, Yuanjie; Jiang, Yadong

    2016-09-01

    An acetylcholinesterase (AChE)-reduced graphene oxide (RGO) hybrid films based biosensor enabled by quartz crystal microbalance (QCM) has been developed for the detection of organophosphorus neurotoxin in gas phase at room temperature. To improve the sensing performance, RGO was used to immobilize large quantities of enzyme and provide a favorable microenvironment to maintain the enzyme activity. The experimental results reveal that the response of AChE-RGO/glutaraldehyde based sensors is about 8 times larger than that of the AChE with the sensitivity of 1.583 Hz/mg/m3. 1.0 mg amount of RGO, 5% concentration of glutaraldehyde and pH 6.8 is the optimal condition of this biosensor.

  12. Highly-substrate active isoenzyme acetylcholinesterase-II, in rosy eye mutant of Aedes aegypti mosquito.

    PubMed

    Mourya, D T; Gokhale, M D; Barde, P V; Deobagkar, D N

    2001-08-01

    Insecticide bioassays were carried out on larvae and adults of rosy eye mutant and wildtype strains of A. aegypti. Both the strains were equally susceptible to DDT, malathion and deltamethrin. Biochemical assays showed an increase in acetylcholinesterase enzyme (AChE) activity in all the stages of mutant strain with both the substrates i.e. acetylthiocholine iodide and S-butyrylthiocholine iodide. However, there was no difference in the percent inhibition of enzyme activity with propoxur in these two strains. Polyacrylamide gel electrophoresis performed in native conditions on the homogenates of adults of rosy eye mosquitoes showed that AChE-II allele was highly active with the substrate acetylthiocholine iodide as compared to wildtype strain. Frequency of the highly active AChE-II allele in the mutant strain was about 68%, whereas it was about 5% in the wildtype strain.

  13. Graphene quantum dots for ultrasensitive detection of acetylcholinesterase and its inhibitors

    NASA Astrophysics Data System (ADS)

    Li, Nan; Wang, Xuewan; Chen, Jie; Sun, Lei; Chen, Peng

    2015-09-01

    Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of novel applications including development of optical sensors. Herein, a GQD-based fluorometric sensor is devised to detect acetylcholinesterase (AChE, a critical enzyme in central nervous system and neuromuscular junctions) with an ultralow detection limit (0.58 pM with S/N of 5.0), using a photoluminescence ‘turn-off’ mechanism. This simple ‘mix-and-detect’ platform can also be employed to sense a variety of compounds that can directly or indirectly inhibit the enzymatic activities of AChE, such as nerve gases, pesticides, and therapeutic drugs. As the proof-of-concept demonstrations, we show the sensitive detection of paraoxon (a pesticide), tacrine (a drug to treat Alzheimer’s disease), and dopamine (an important neurotransmitter).

  14. Ethynylphenyl carbonates and carbamates as dual-action acetylcholinesterase inhibitors and anti-inflammatory agents.

    PubMed

    Saxena, Jaya; Meloni, David; Huang, Mou-Tuan; Heck, Diane E; Laskin, Jeffrey D; Heindel, Ned D; Young, Sherri C

    2015-12-01

    Novel ethynylphenyl carbonates and carbamates containing carbon- and silicon-based choline mimics were synthesized from their respective phenol and aniline precursors and screened for anticholinesterase and anti-inflammatory activities. All molecules were micromolar inhibitors of acetylcholinesterase (AChE), with IC50s of 28-86 μM; the carbamates were two-fold more potent than the carbonates. Two of the most potent AChE inhibitors suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation by 40%. Furthermore, these molecules have physicochemical properties in the range of other CNS drugs. These molecules have the potential to treat inflammation; they could also dually target Alzheimer's disease through restoration of cholinergic balance and inflammation suppression.

  15. Development of ESI-MS-based continuous enzymatic assay for real-time monitoring of enzymatic reactions of acetylcholinesterase.

    PubMed

    Fu, Qiang; Tang, Jun; Cui, Meng; Zheng, Zhong; Liu, Zhiqiang; Liu, Shuying

    2015-05-15

    The continuous enzymatic assay based on ESI-MS was developed to real-time monitoring of enzymatic reactions of acetylcholinesterase (AChE). The changes of product concentrations were continuously measured. Calibration curves were established for quantitative calculation. By this method, the Michaelis constant (Km) of acetylcholinesterase was determined to be 70.60±0.93μM and Huperzine A as an effective inhibitor of acetylcholinesterase displayed a mixed inhibition with competitive and noncompetitive inhibition behaviors. The half maximal inhibitory concentration (IC50) and inhibition constant (Ki) value of Huperzine A were also calculated as 48.51±1.16nM and 26.73±0.27nM, respectively. This method provides the rapid and accurate ways to monitor enzyme reactions.

  16. Acetylcholinesterase inhibition within the lycorine series of Amaryllidaceae alkaloids.

    PubMed

    Nair, Jerald J; van Staden, Johannes

    2012-07-01

    The plant family Amaryllidaceae occupies a privileged status within the botanical hierarchy due to its horticultural and ornamental appeal, as well as its widespread usage in the traditional medicinal practices of indigenous peoples across the globe. Of greater significance are the unique, structurally-diverse alkaloid constituents produced by members of the family, which has spawned several biologically significant molecules. In this regard, the Alzheimer's drug galanthamine has gained much prominence due to its selective and reversible inhibitory interaction with the enzyme acetylcholinesterase (AChE), of significance in the progression of neurodegeneration associated with Alzheimer's disease (AD). The lycorine series of compounds within the family have recently emerged as novel inhibitors of AChE, in some instances with higher levels of activity compared with the commercial drug galanthamine, making them attractive targets for natural product and synthetically-driven structure-activity relationship studies. This brief survey traces the emergence of lycorine compounds over the past decade as promising leads in the therapeutic approach towards AD and their possible future advancement onto the clinical stage.

  17. A 1-methyl-4-piperidinyl cytectrene carboxylate labeled by the technetium 99m, a radiotracer for rat brain acetylcholinesterase activity.

    PubMed

    Mejri, Najoua; Barhoumi, Chokri; Trabelsi, Moez; Mekni, Abdelkader; Said, Nadia Malek; Saidi, Mouldi

    2010-02-01

    Alzheimer's disease (AD) is a degenerative neurological disorder that causes progressive and irreversible loss of connections between brain cells and loss of mental functions. Clinical and postmortem studies show that the biochemical changes in brains of AD patients include decrease in acetylcholinesterase (AChE) activity. Our aim was to study AChE activity using piperidinyl ester labelled with technetium-99m. In vivo and in vitro studies demonstrated that labelled piperidinyl ester was a substrate for AChE. The hydrolytic rate of this substrate was measured and the specificity was evaluated using the inhibitor BW284c51. The rhenium analogues of the technetium-labelled substrate were used to determine the affinity constant (K(m)) and the maximum reaction velocity (V(max)) because of the high specific activity of technetium. The high hydrolytic rate and high specificity of the substrate for AChE make it suitable as an in vivo radiotracer for studying AChE activity in the brain.

  18. Designing Second Generation Anti-Alzheimer Compounds as Inhibitors of Human Acetylcholinesterase: Computational Screening of Synthetic Molecules and Dietary Phytochemicals

    PubMed Central

    Amat-ur-Rasool, Hafsa; Ahmed, Mehboob

    2015-01-01

    Alzheimer's disease (AD), a big cause of memory loss, is a progressive neurodegenerative disorder. The disease leads to irreversible loss of neurons that result in reduced level of acetylcholine neurotransmitter (ACh). The reduction of ACh level impairs brain functioning. One aspect of AD therapy is to maintain ACh level up to a safe limit, by blocking acetylcholinesterase (AChE), an enzyme that is naturally responsible for its degradation. This research presents an in-silico screening and designing of hAChE inhibitors as potential anti-Alzheimer drugs. Molecular docking results of the database retrieved (synthetic chemicals and dietary phytochemicals) and self-drawn ligands were compared with Food and Drug Administration (FDA) approved drugs against AD as controls. Furthermore, computational ADME studies were performed on the hits to assess their safety. Human AChE was found to be most approptiate target site as compared to commonly used Torpedo AChE. Among the tested dietry phytochemicals, berberastine, berberine, yohimbine, sanguinarine, elemol and naringenin are the worth mentioning phytochemicals as potential anti-Alzheimer drugs The synthetic leads were mostly dual binding site inhibitors with two binding subunits linked by a carbon chain i.e. second generation AD drugs. Fifteen new heterodimers were designed that were computationally more efficient inhibitors than previously reported compounds. Using computational methods, compounds present in online chemical databases can be screened to design more efficient and safer drugs against cognitive symptoms of AD. PMID:26325402

  19. An optical fiber biosensor for chlorpyrifos using a single sol-gel film containing acetylcholinesterase and bromothymol blue.

    PubMed

    Kuswandi, Bambang; Fikriyah, Chulaifah Indah; Gani, Agus Abdul

    2008-01-15

    An optical fiber biosensor consisting of acetylcholinesterase (AChE) and bromothymol blue (BTB) doped sol-gel film was employed to detect organophosphate pesticide chlorpyrifos. The main advantage of this optical biosensor is the use of a single sol-gel film with immobilized AChE and BTB. The compatibility of this mixture (AChE and BTB) with the sol-gel matrix has prevented leaching of the film. The immobilization of the enzyme and indicator was simple without chemical modification. The biosensing element on single sol-gel film has been placed inside the flow-cell for flow system. In the presence of a constant AChE, a color change of the BTB and the measured reflected signal at wavelength 622nm could be related to the pesticide concentration in the sample solutions. The performance of optical biosensor in the flow system has been optimized, including chemical and physical parameters. The response time of the biosensor is 8min. A linear calibration curve of chlorpyrifos against the percentage inhibition of AChE was obtained from 0.05 to 2.0mg/L of chlorpyrifos (18-80% inhibition, R(2)=0.9869, n=6). The detection limit for chlorpyrifos was 0.04mg/L. The results of the analysis of 0.5-1.5mg/L of chlorpyrifos using this optical biosensor agreed well with chromatographic method.

  20. A Novel Application of Multiscale Entropy in Electroencephalography to Predict the Efficacy of Acetylcholinesterase Inhibitor in Alzheimer's Disease

    PubMed Central

    Tsai, Ping-Huang; Chang, Shih-Chieh; Liu, Fang-Chun; Tsao, Jenho; Wang, Yung-Hung; Lo, Men-Tzung

    2015-01-01

    Alzheimer's disease (AD) is the most common form of dementia. According to one hypothesis, AD is caused by the reduced synthesis of the neurotransmitter acetylcholine. Therefore, acetylcholinesterase (AChE) inhibitors are considered to be an effective therapy. For clinicians, however, AChE inhibitors are not a predictable treatment for individual patients. We aimed to disclose the difference by biosignal processing. In this study, we used multiscale entropy (MSE) analysis, which can disclose the embedded information in different time scales, in electroencephalography (EEG), in an attempt to predict the efficacy of AChE inhibitors. Seventeen newly diagnosed AD patients were enrolled, with an initial minimental state examination (MMSE) score of 18.8 ± 4.5. After 12 months of AChE inhibitor therapy, 7 patients were responsive and 10 patients were nonresponsive. The major difference between these two groups is Slope 2 (MSE6 to 20). The area below the receiver operating characteristic (ROC) curve of Slope 2 is 0.871 (95% CI = 0.69–1). The sensitivity is 85.7% and the specificity is 60%, whereas the cut-off value of Slope 2 is −0.024. Therefore, MSE analysis of EEG signals, especially Slope 2, provides a potential tool for predicting the efficacy of AChE inhibitors prior to therapy. PMID:26120358

  1. Exploring the physicochemical properties of oxime-reactivation therapeutics for cyclosarin, sarin, tabun, and VX inactivated acetylcholinesterase.

    PubMed

    Esposito, Emilio Xavier; Stouch, Terry R; Wymore, Troy; Madura, Jeffry D

    2014-01-21

    The inactivation of acetylcholinesterase (AChE) by organophosphorus agent (OP) compounds is a serious problem regardless of how the individual was exposed. The reactivation of OP-inactivated AChE is dependent on the OP conjugate, and commonly a specific oxime is better at reactivating a specific OP conjugate than several diverse OP conjugates. The presented research explores the physicochemical properties needed for the reactivation of OP-inactivated AChE. Four different OPs, cyclosarin, sarin, tabun, and VX, were analyzed using the same set of oxime reactivators. A trial descriptor pool of semiempirical, traditional, and molecular interaction field descriptors was used to construct an ensemble of QSAR models for each OP-conjugate pair. Based on the molecular information and the cross-validation ability, individual QSAR models were selected to be part of an OP-conjugate consensus model. The OP-conjugate specific models provide important insight into the physicochemical properties required to reactivate the OP conjugates of interest. The reactivation of AChE inactivated with either cyclosarin or tabun requires the oxime therapeutic to possess an overall polar-positive surface area. Oxime therapeutics for the reactivation of sarin-inactivated AChE are conformationally dependent while oxime reverse therapeutics for VX require a compact region with a highly hydrophilic region and two positively charged pyridine rings.

  2. Reactivation of organophosphate-inhibited human, Cynomolgus monkey, swine and guinea pig acetylcholinesterase by MMB-4: A modified kinetic approach

    SciTech Connect

    Worek, Franz; Wille, Timo; Aurbek, Nadine; Eyer, Peter; Thiermann, Horst

    2010-12-15

    Treatment of poisoning by highly toxic organophosphorus compounds (OP, nerve agents) is a continuous challenge. Standard treatment with atropine and a clinically used oxime, obidoxime or pralidoxime is inadequate against various nerve agents. For ethical reasons testing of oxime efficacy has to be performed in animals. Now, it was tempting to investigate the reactivation kinetics of MMB-4, a candidate oxime to replace pralidoxime, with nerve agent-inhibited acetylcholinesterase (AChE) from human and animal origin in order to provide a kinetic basis for the proper assessment of in vivo data. By applying a modified kinetic approach, allowing the use of necessary high MMB-4 concentrations, it was possible to determine the reactivation constants with sarin-, cyclosarin-, VX-, VR- and tabun-inhibited AChE. MMB-4 exhibited a high reactivity and low affinity towards OP-inhibited AChE, except of tabun-inhibited enzyme where MMB-4 had an extremely low reactivity. Species differences between human and animal AChE were low (Cynomolgus) to moderate (swine, guinea pig). Due to the high reactivity of MMB-4 a rapid reactivation of inhibited AChE can be anticipated at adequate oxime concentrations which are substantially higher compared to HI-6. Additional studies are necessary to determine the in vivo toxicity, tolerability and pharmacokinetics of MMB-4 in humans in order to enable a proper assessment of the value of this oxime as an antidote against nerve agent poisoning.

  3. Designing Second Generation Anti-Alzheimer Compounds as Inhibitors of Human Acetylcholinesterase: Computational Screening of Synthetic Molecules and Dietary Phytochemicals.

    PubMed

    Amat-Ur-Rasool, Hafsa; Ahmed, Mehboob

    2015-01-01

    Alzheimer's disease (AD), a big cause of memory loss, is a progressive neurodegenerative disorder. The disease leads to irreversible loss of neurons that result in reduced level of acetylcholine neurotransmitter (ACh). The reduction of ACh level impairs brain functioning. One aspect of AD therapy is to maintain ACh level up to a safe limit, by blocking acetylcholinesterase (AChE), an enzyme that is naturally responsible for its degradation. This research presents an in-silico screening and designing of hAChE inhibitors as potential anti-Alzheimer drugs. Molecular docking results of the database retrieved (synthetic chemicals and dietary phytochemicals) and self-drawn ligands were compared with Food and Drug Administration (FDA) approved drugs against AD as controls. Furthermore, computational ADME studies were performed on the hits to assess their safety. Human AChE was found to be most approptiate target site as compared to commonly used Torpedo AChE. Among the tested dietry phytochemicals, berberastine, berberine, yohimbine, sanguinarine, elemol and naringenin are the worth mentioning phytochemicals as potential anti-Alzheimer drugs The synthetic leads were mostly dual binding site inhibitors with two binding subunits linked by a carbon chain i.e. second generation AD drugs. Fifteen new heterodimers were designed that were computationally more efficient inhibitors than previously reported compounds. Using computational methods, compounds present in online chemical databases can be screened to design more efficient and safer drugs against cognitive symptoms of AD.

  4. Electronic structure calculations toward new potentially AChE inhibitors

    NASA Astrophysics Data System (ADS)

    de Paula, A. A. N.; Martins, J. B. L.; Gargano, R.; dos Santos, M. L.; Romeiro, L. A. S.

    2007-10-01

    The main purpose of this study was the use of natural non-isoprenoid phenolic lipid of cashew nut shell liquid from Anacardium occidentale as lead material for generating new potentially candidates of acetylcholinesterase inhibitors. Therefore, we studied the electronic structure of 15 molecules derivatives from the cardanol using the following groups: methyl, acetyl, N, N-dimethylcarbamoyl, N, N-dimethylamine, N, N-diethylamine, piperidine, pyrrolidine, and N-benzylamine. The calculations were performed at RHF level using 6-31G, 6-31G(d), 6-31+G(d) and 6-311G(d,p) basis functions. Among the proposed compounds we found that the structures with substitution by acetyl, N, N-dimethylcarbamoyl, N, N-dimethylamine, and pyrrolidine groups were better correlated to rivastigmine indicating possible activity.

  5. Screening the methanol extracts of some Iranian plants for acetylcholinesterase inhibitory activity

    PubMed Central

    Gholamhoseinian, A.; Moradi, M.N.; Sharifi-far, F.

    2009-01-01

    Acetylcholinesterase (AChE) is the main enzyme for the breakdown of acetylcholine. Nowadays, usage of the inhibitors of this enzyme is one of the most important types of treatment of mild to moderate neurodegenerative diseases such as Alzheimer’s disease. Herbal medicines can be a new source of inhibitors of this enzyme. In this study we examined around 100 different plants to evaluate their inhibitory properties for AChE enzyme. Plants were scientifically identified and their extracts were prepared by methanol percolation. Acetylcholinesterase activity was measured using a colorimetric method in the presence or absence of the extracts. Eserine was used as a positive control. Methanol extracts of the Levisticum officinale, Bergeris integrima and Rheum ribes showed more than 50% AChE inhibitory activity. The inhibition kinetics were studied in the presence of the most effective extracts. L. officinale and B. integrima inhibited AChE activity in a non-competitive manner, while R. ribes competitively inhibitied the enzyme as revealed by double-reciprocal Linweaver-Burk plot analysis. Under controlled condition, Km and Vmax values of the enzyme were found to be 9.4 mM and 0.238 mM/min, respectively. However, in the presence of L. officinale, B. integrima, and R. ribes extracts, Vmax values were 0.192, 0.074 and 0.238 mM/min, respectively. Due to the competitive inhibition of the enzyme by R. ribes extract, the Km value of 21.2 mM was obtained. The concentration required for 50% enzyme inhibition (IC50 value) was 0.5, 0.9, and 0.95 mg/ml for the L. officinale, B. integrima and R. ribes extracts, respectively. The IC50 of the eserine was determined to be 0.8 mg/ml. PMID:21589805

  6. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  7. Docking of the alkaloid geissospermine into acetylcholinesterase: a natural scaffold targeting the treatment of Alzheimer's disease.

    PubMed

    Araújo, Jocley Queiroz; Lima, Josélia Alencar; Pinto, Angelo da Cunha; de Alencastro, Ricardo Bicca; Albuquerque, Magaly Girão

    2011-06-01

    Pharmacological studies from our group [Lima et al. Pharmacol Biochem Behav 92:508, (2009)] revealed that geissospermine (GSP), the major alkaloid of the bark extract of Brazilian Geissospermum vellosii, inhibits acetylcholinesterases (AChEs) in the brains of rats and electric eels (Electrophorus electricus). However, the binding mode (i.e., conformation and orientation) of this indole-indoline alkaloid into the AChE active site is unknown. Therefore, in order to propose a plausible binding mode between GSP and AChE, which might explain the observed experimental inhibitory activity, we performed comparative automatic molecular docking simulations using the AutoDock and Molegro Virtual Docker (MVD) programs. A sample of ten crystal structures of the Pacific electric ray (Torpedo californica) TcAChE, in complex with ten diverse active site ligands, was selected as a robust re-docking validation test, and also for GSP docking. The MVD results indicate a preferential binding mode between GSP and AChE, in which GSP functional groups may perform specific interactions with residues in the enzyme active site, according to the ligand-protein contacts detected by the LPC/CSU server. Four hydrogen bonds were detected between GSP and Tyr121, Ser122, Ser200, and His440, in which the last two residues belong to the catalytic triad (Ser200···His440···Glu327). Hydrophobic and π-π stacking interactions were also detected between GSP and Phe330 and Trp84, respectively; these are involved in substrate stabilization at the active site. This study provides the basis to propose structural changes to the GSP structure, such as molecular simplification and isosteric replacement, in order to aid the design of new potential AChE inhibitors that are relevant to the treatment of Alzheimer's disease.

  8. The discovery of potential acetylcholinesterase inhibitors: A combination of pharmacophore modeling, virtual screening, and molecular docking studies

    PubMed Central

    2011-01-01

    Background Alzheimer's disease (AD) is the most common cause of dementia characterized by progressive cognitive impairment in the elderly people. The most dramatic abnormalities are those of the cholinergic system. Acetylcholinesterase (AChE) plays a key role in the regulation of the cholinergic system, and hence, inhibition of AChE has emerged as one of the most promising strategies for the treatment of AD. Methods In this study, we suggest a workflow for the identification and prioritization of potential compounds targeted against AChE. In order to elucidate the essential structural features for AChE, three-dimensional pharmacophore models were constructed using Discovery Studio 2.5.5 (DS 2.5.5) program based on a set of known AChE inhibitors. Results The best five-features pharmacophore model, which includes one hydrogen bond donor and four hydrophobic features, was generated from a training set of 62 compounds that yielded a correlation coefficient of R = 0.851 and a high prediction of fit values for a set of 26 test molecules with a correlation of R2 = 0.830. Our pharmacophore model also has a high Güner-Henry score and enrichment factor. Virtual screening performed on the NCI database obtained new inhibitors which have the potential to inhibit AChE and to protect neurons from Aβ toxicity. The hit compounds were subsequently subjected to molecular docking and evaluated by consensus scoring function, which resulted in 9 compounds with high pharmacophore fit values and predicted biological activity scores. These compounds showed interactions with important residues at the active site. Conclusions The information gained from this study may assist in the discovery of potential AChE inhibitors that are highly selective for its dual binding sites. PMID:21251245

  9. Morphinans and isoquinolines: acetylcholinesterase inhibition, pharmacophore modeling, and interaction with opioid receptors.

    PubMed

    Schuster, Daniela; Spetea, Mariana; Music, Melisa; Rief, Silvia; Fink, Monika; Kirchmair, Johannes; Schütz, Johannes; Wolber, Gerhard; Langer, Thierry; Stuppner, Hermann; Schmidhammer, Helmut; Rollinger, Judith M

    2010-07-15

    Following indications from pharmacophore-based virtual screening of natural product databases, morphinan and isoquinoline compounds were tested in vitro for acetylcholinesterase (AChE) inhibition. After the first screen, active and inactive compounds were used to build a ligand-based pharmacophore model in order to prioritize compounds for biological testing. Among the virtual hits tested, the enrichment of actives was significantly higher than in a random selection of test compounds. The most active compounds were biochemically tested for their activity on mu, delta, and kappa opioid receptors.

  10. Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

    PubMed Central

    Berman, Jonathan Dembitz; Young, Michael

    1971-01-01

    Affinity chromatography has been used to purify acetylcholinesterase both from the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes. For this purpose, several specific enzymic inhibitors of each protein were synthesized and joined covalently to an insoluble support resin. AchE is selectively retained by such inhibitor-resins when highly impure solutions are chromatographed upon them. After removal from the resin, both enzymes are electrophoretically homogeneous and they may be recovered in yields of 75% or more. Images PMID:5277092

  11. Distribution of Intravenously Administered Acetylcholinesterase Inhibitor and Acetylcholinesterase Activity in the Adrenal Gland: 11C-Donepezil PET Study in the Normal Rat

    PubMed Central

    Watabe, Tadashi; Naka, Sadahiro; Ikeda, Hayato; Horitsugi, Genki; Kanai, Yasukazu; Isohashi, Kayako; Ishibashi, Mana; Kato, Hiroki; Shimosegawa, Eku; Watabe, Hiroshi; Hatazawa, Jun

    2014-01-01

    Purpose Acetylcholinesterase (AChE) inhibitors have been used for patients with Alzheimer's disease. However, its pharmacokinetics in non-target organs other than the brain has not been clarified yet. The purpose of this study was to evaluate the relationship between the whole-body distribution of intravenously administered 11C-Donepezil (DNP) and the AChE activity in the normal rat, with special focus on the adrenal glands. Methods The distribution of 11C-DNP was investigated by PET/CT in 6 normal male Wistar rats (8 weeks old, body weight  = 220±8.9 g). A 30-min dynamic scan was started simultaneously with an intravenous bolus injection of 11C-DNP (45.0±10.7 MBq). The whole-body distribution of the 11C-DNP PET was evaluated based on the Vt (total distribution volume) by Logan-plot analysis. A fluorometric assay was performed to quantify the AChE activity in homogenized tissue solutions of the major organs. Results The PET analysis using Vt showed that the adrenal glands had the 2nd highest level of 11C-DNP in the body (following the liver) (13.33±1.08 and 19.43±1.29 ml/cm3, respectively), indicating that the distribution of 11C-DNP was the highest in the adrenal glands, except for that in the excretory organs. The AChE activity was the third highest in the adrenal glands (following the small intestine and the stomach) (24.9±1.6, 83.1±3.0, and 38.5±8.1 mU/mg, respectively), indicating high activity of AChE in the adrenal glands. Conclusions We demonstrated the whole-body distribution of 11C-DNP by PET and the AChE activity in the major organs by fluorometric assay in the normal rat. High accumulation of 11C-DNP was observed in the adrenal glands, which suggested the risk of enhanced cholinergic synaptic transmission by the use of AChE inhibitors. PMID:25225806

  12. Mutations in Acetylcholinesterase2 (ace2) increase the insensitivity of acetylcholinesterase to fosthiazate in the root-knot nematode Meloidogyne incognita.

    PubMed

    Huang, Wen-Kun; Wu, Qin-Song; Peng, Huan; Kong, Ling-An; Liu, Shi-Ming; Yin, Hua-Qun; Cui, Ru-Qiang; Zhan, Li-Ping; Cui, Jiang-Kuan; Peng, De-Liang

    2016-11-29

    The root-knot nematode Meloidogyne incognita causes severe damage to continuously cropping vegetables. The control of this nematode relies heavily on organophosphate nematicides in China. Here, we described resistance to the organophosphate nematicide fosthiazate in a greenhouse-collected resistant population (RP) and a laboratory susceptible population (SP) of M. incognita. Fosthiazate was 2.74-fold less toxic to nematodes from RP than that from SP. Quantitative real-time PCR revealed that the acetylcholinesterase2 (ace2) transcription level in the RP was significantly higher than that in the SP. Eighteen nonsynonymous amino acid differences in ace2 were observed between the cDNA fragments of the RP and SP. The acetylcholinesterase (AChE) protein activity in the RP was significantly reduced compared with that in the SP. After knocking down the ace2 gene, the ace2 transcription level was significantly decreased, but no negative impact on the infection of juveniles was observed. The 50% lethal concentration of the RNAi RP population decreased 40%, but the inhibition rate of fosthiazate against AChE activity was significantly increased in RP population. Thus, the increased fosthiazate insensitivity in the M. incognita resistant population was strongly associated with mutations in ace2. These results provide valuable insights into the resistance mechanism of root-knot nematode to organophosphate nematicides.

  13. Mutations in Acetylcholinesterase2 (ace2) increase the insensitivity of acetylcholinesterase to fosthiazate in the root-knot nematode Meloidogyne incognita

    PubMed Central

    Huang, Wen-Kun; Wu, Qin-Song; Peng, Huan; Kong, Ling-An; Liu, Shi-Ming; Yin, Hua-Qun; Cui, Ru-Qiang; Zhan, Li-Ping; Cui, Jiang-Kuan; Peng, De-Liang

    2016-01-01

    The root-knot nematode Meloidogyne incognita causes severe damage to continuously cropping vegetables. The control of this nematode relies heavily on organophosphate nematicides in China. Here, we described resistance to the organophosphate nematicide fosthiazate in a greenhouse-collected resistant population (RP) and a laboratory susceptible population (SP) of M. incognita. Fosthiazate was 2.74-fold less toxic to nematodes from RP than that from SP. Quantitative real-time PCR revealed that the acetylcholinesterase2 (ace2) transcription level in the RP was significantly higher than that in the SP. Eighteen nonsynonymous amino acid differences in ace2 were observed between the cDNA fragments of the RP and SP. The acetylcholinesterase (AChE) protein activity in the RP was significantly reduced compared with that in the SP. After knocking down the ace2 gene, the ace2 transcription level was significantly decreased, but no negative impact on the infection of juveniles was observed. The 50% lethal concentration of the RNAi RP population decreased 40%, but the inhibition rate of fosthiazate against AChE activity was significantly increased in RP population. Thus, the increased fosthiazate insensitivity in the M. incognita resistant population was strongly associated with mutations in ace2. These results provide valuable insights into the resistance mechanism of root-knot nematode to organophosphate nematicides. PMID:27897265

  14. Novel nerve-agent antidote design based on crystallographic and mass spectrometric analyses of tabun-conjugated acetylcholinesterase in complex with antidotes.

    PubMed

    Ekström, F J; Astot, C; Pang, Y-P

    2007-09-01

    Organophosphorus compound-based nerve agents inhibit the essential enzyme acetylcholinesterase (AChE) causing acute toxicity and death. Clinical treatment of nerve-agent poisoning is to use oxime-based antidotes to reactivate the inhibited AChE. However, the nerve agent tabun is resistant to oximes. To design improved oximes, crystal structures of a tabun-conjugated AChE in complex with different oximes are needed to guide the structural modifications of known antidotes. However, this type of structure is extremely challenging to obtain because both deamidation of the tabun conjugate and reactivation of AChE occur during crystallographic experiments. Here we report, for the first time, the crystal structures of Ortho-7 and HLö-7 in complex with AChE that is conjugated to an intact tabun. These structures were determined by our new strategy of combining crystallographic and mass spectrometric analyses of AChE crystals. The results explain the relative reactivation potencies of the two oximes and offer insights into improving known medical antidotes.

  15. Multiple Mutations on the Second Acetylcholinesterase Gene Associated With Dimethoate Resistance in the Melon Aphid, Aphis gossypii (Hemiptera: Aphididae).

    PubMed

    Lokeshwari, D; Krishna Kumar, N K; Manjunatha, H

    2016-04-01

    The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is an important cosmopolitan and extremely polyphagous species capable of causing direct and indirect damage to various crops. Insecticide resistance in melon aphids is of particular concern. To determine the basis of resistance, organophosphate (OP)-resistant strains of A. gossypii were obtained by continuous selection with dimethoate in the laboratory, and resistance mechanisms were investigated along with susceptible strains. Three resistant strains LKR-1, LKR-2, and LKR-3 exhibiting 270-, 243-, and 210-fold resistance obtained after 30 generations of selection with dimethoate, respectively, were utilized in this study. The role of acetylcholinesterase (AChE), a target enzyme for OPs and carbamates (CMs), was investigated. AChE enzyme assay revealed that there was no significant change in the activities of AChE in resistant and susceptible strains. However, AChE inhibitory assay showed that 50% of the enzyme activity in resistant strains was inhibited at significantly higher concentration of dimethoate (131.87, 158.65, and 99.29 µmolL(−1)) as compared with susceptible strains (1.75 and 2.01 µmolL(−1)), indicating AChE insensitivity owing to altered AChE. Molecular diagnostic tool polymerase chain reaction-restriction fragment length polymorphism revealed the existence of two consistent non-synonymous point mutations, single-nucleotide polymorphism, viz., A302S (equivalent to A201 in Torpedo californica Ayres) and S431F (equivalent to F331 in T. californica), in the AChE gene Ace2 of resistant strains. Further, cloning and sequencing of a partial fragment of Ace2 (897 bp) gene from susceptible and resistant strains revealed an additional novel mutation G221A in resistant strains, LKR-1 and LKR-2. Susceptible Ace2 genes shared 99.6 and 98.9% identity at the nucleic acid and amino acid levels with resistant ones, respectively. Functional analysis of these point mutations was assessed by in

  16. Immobilization of Acetylcholinesterase on Screen-Printed Electrodes. Application to the Determination of Arsenic(III)

    PubMed Central

    Sanllorente-Méndez, Silvia; Domínguez-Renedo, Olga; Arcos-Martínez, M. Julia

    2010-01-01

    Enzymatic amperometric procedures for measuring arsenic, based on the inhibitive action of this metal on acetylcholinesterase enzyme activity, have been developed. Screen-printed carbon electrodes (SPCEs) were used with acetylcholinesterase covalently bonded directly to its surface. The amperometric response of acetylcholinesterase was affected by the presence of arsenic ions, which caused a decrease in the current intensity. The experimental optimum working conditions of pH, substrate concentration and potential applied, were established. Under these conditions, repeatability and reproducibility of biosensors were determined, reaching values below 4% in terms of relative standard deviation. The detection limit obtained for arsenic was 1.1 × 10−8 M for Ach/SPCE biosensor. Analysis of the possible effect of the presence of foreign ions in the solution was performed. The method was applied to determine levels of arsenic in spiked tap water samples. PMID:22294918

  17. Structure-activity relationships and binding mode in the human acetylcholinesterase active site of pseudo-irreversible inhibitors related to xanthostigmine.

    PubMed

    Rizzo, Stefano; Cavalli, Andrea; Ceccarini, Luisa; Bartolini, Manuela; Belluti, Federica; Bisi, Alessandra; Andrisano, Vincenza; Recanatini, Maurizio; Rampa, Angela

    2009-04-01

    Structure-activity relationship studies on acetylcholinesterase (AChE) inhibitors were extended to newly synthesized compounds derived from the lead compound xantostigmine (1). The xanthone ring of compound 1 was replaced with several different scaffolds based on the benzopyran skeleton, linked to the tertiary amino nitrogen through an heptyloxy chain. These modifications resulted in 19 new compounds, most of them showing activity in the nanomolar-subnanomolar range. Docking and molecular dynamics simulations were carried out to both define a new computational protocol for the simulation of pseudo-irreversibile AChE covalent inhibitors, and to acquire a better understanding of the structure-activity relationships of the present series of compounds. The results of this computational work prompted us to to evaluate the ability of compounds 5 and 13 to inhibit acetylcholinesterase-induced Abeta aggregation.

  18. HI-6 assisted catalytic scavenging of VX by acetylcholinesterase choline binding site mutants.

    PubMed

    Maček Hrvat, Nikolina; Žunec, Suzana; Taylor, Palmer; Radić, Zoran; Kovarik, Zrinka

    2016-11-25

    The high toxicity of organophosphorus compounds originates from covalent inhibition of acetylcholinesterase (AChE), an essential enzyme in cholinergic neurotransmission. Poisonings that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OPs focuses on the use of bioscavengers against the parent organophosphate. Our previous research showed that AChE mutagenesis can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates, while dramatically slowing down rates of OP-conjugate dealkylation (aging). Herein, we demonstrate an efficient HI-6-assisted VX detoxification, both ex vivo in human blood and in vivo in mice by hAChE mutants modified at the choline binding site (Y337A and Y337A/F338A). The catalytic scavenging of VX in mice improved therapeutic outcomes preventing lethality and resulted in a delayed onset of toxicity symptoms.

  19. Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2.

    PubMed

    Kafurke, Uwe; Erijman, Ariel; Aizner, Yonatan; Shifman, Julia M; Eichler, Jutta

    2015-09-01

    Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein-ligand interactions. Such mimics of protein binding sites can currently be generated through structure-based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin-2 (Fas) because the structure and function of this complex is well understood. Structure-based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE-specific peptide-Fas interaction.

  20. Expression and characterization of recombinant Locusta migratoria manilensis acetylcholinesterase 1 in Pichia pastoris.

    PubMed

    Zhou, Xiaoxia; Xia, Yuxian

    2011-05-01

    The acetylcholinesterase 1 from Locusta migratoria manilensis (LmAChE1) was successfully expressed in methylotrophic yeast Pichia pastoris KM71. The maximum expression of recombinant LmAChE1 (reLmAChE1) was achieved after 9 days of induction at 2.5% methanol. The reLmAChE1 was first precipitated with ammonium sulfate (50% saturation) and then was purified with nickel affinity chromatography. The enzyme was purified 3.2×10(3)-fold with a yield of 68% and a specific activity of 8.1 U/mg. The purified reLmAChE1 exhibited highest activity at 30°C in 100 mM phosphate buffer (pH 7.4), and its activity could be inhibited by eserine sulfate and pentan-3-one-dibromide (BW284c51). Substrate specificity analysis showed that the purified reLmAChE1 preferred acetylthiocholine (ATC) and propionylthiocholine (BTC) rather than butyrylthiocholine (BTC). When ATC was used as substrate, the K(m) and V(max) values for the reLmAChE1 were 24.8 μM and 9.5 μmol/min/mg, respectively.

  1. 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

  2. Determination of AChE levels and genotoxic effects in farmers occupationally exposed to pesticides.

    PubMed

    Naravaneni, Rambabu; Jamil, Kaiser

    2007-09-01

    Pesticides can cause cytogenetic effects and lower the acetyl cholinesterase (AChE) levels in farmers exposed to pesticides. In this study, 210 farmers exposed to pesticides and 160 non-exposed individuals were enrolled for determining the genotoxicity and AChE levels. The AChE levels were determined in plasma and RBC lysate from blood samples collected from farmers and control subjects. AChE (true and pseudo) estimation done by the colorimetric method revealed that there was a progressive fall in both the RBC and plasma AChE levels in exposed individuals compared to unexposed individuals, which correlated with the severity of exposure (253.5 versus 311.1 and 142.3 versus 152.1; P < 0.001). Cytogenetic studies showed an increase in DNA damage and higher chromosomal aberrations (CAs) in exposed farmers compared to the control subjects (26.13 versus 07.61 and 21.37 versus 1.52; P < 0.001). When comparing the AChE levels with DNA damage and structural CA frequencies, there was a negative linear correlation. Therefore based on these findings, it is concluded that genotoxic biomarkers like CA frequencies, DNA damage data along with AChE levels are important parameters for determining farmer's health who are exposed to pesticides in any situation.

  3. Acetylcholinesterase Activity, Cohabitation with Floricultural Workers, and Blood Pressure in Ecuadorian Children

    PubMed Central

    Jacobs, David R.; Himes, John H.; Alexander, Bruce H.

    2013-01-01

    Background: Acetylcholinesterase (AChE) inhibitors are commonly used pesticides that can effect hemodynamic changes through increased cholinergic stimulation. Children of agricultural workers are likely to have paraoccupational exposures to pesticides, but the potential physiological impact of such exposures is unclear. Objectives: We investigated whether secondary pesticide exposures were associated with blood pressure and heart rate among children living in agricultural Ecuadorian communities. Methods: This cross-sectional study included 271 children 4–9 years of age [51% cohabited with one or more flower plantation workers (mean duration, 5.2 years)]. Erythrocyte AChE activity was measured using the EQM Test-mate system. Linear regression models were used to estimate associations of systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate with AChE activity, living with flower workers, duration of cohabitation with a flower worker, number of flower workers in the child’s home, and number of practices that might increase children’s exposure to pesticides. Results: Mean (± SD) AChE activity was 3.14 ± 0.49 U/mL. A 1-U/mL decrease in AChE activity was associated with a 2.86-mmHg decrease in SBP (95% CI: –5.20, –0.53) and a 2.89-mmHg decrease in DBP (95% CI: –5.00, –0.78), after adjustment for potential confounders. Children living with flower workers had lower SBP (–1.72 mmHg; 95% CI: –3.53, 0.08) than other children, and practices that might increase exposure also were associated with lower SBP. No significant associations were found between exposures and heart rate. Conclusions: Our findings suggest that subclinical secondary exposures to pesticides may affect vascular reactivity in children. Additional research is needed to confirm these findings. PMID:23359481

  4. The effect of aspartame metabolites on human erythrocyte membrane acetylcholinesterase activity.

    PubMed

    Tsakiris, Stylianos; Giannoulia-Karantana, Aglaia; Simintzi, Irene; Schulpis, Kleopatra H

    2006-01-01

    Studies have implicated aspartame (ASP) with neurological problems. The aim of this study was to evaluate acetylcholinesterase (AChE) activity in human erythrocyte membranes after incubation with the sum of ASP metabolites, phenylalanine (Phe), methanol (met) and aspartic acid (aspt), or with each one separately. Erythrocyte membranes were obtained from 12 healthy individuals and were incubated with ASP hydrolysis products for 1 h at 37 degrees C. AChE was measured spectrophotometrically. Incubation of membranes with ASP metabolites corresponding with 34 mg/kg, 150 mg/kg or 200 mg/kg of ASP consumption resulted in an enzyme activity reduction by -33%, -41%, and -57%, respectively. Met concentrations 0.14 mM, 0.60 mM, and 0.80 mM decreased the enzyme activity by -20%, -32% or -40%, respectively. Aspt concentrations 2.80 mM, 7.60 mM or 10.0 mM inhibited membrane AChE activity by -20%, -35%, and -47%, respectively. Phe concentrations 0.14 mM, 0.35 mM or 0.50mM reduced the enzyme activity by -11%, -33%, and -35%, respectively. Aspt or Phe concentrations 0.82 mM or 0.07 mM, respectively, did not alter the membrane AChE activity. It is concluded that low concentrations of ASP metabolites had no effect on the membrane enzyme activity, whereas high or toxic concentrations partially or remarkably decreased the membrane AChE activity, respectively. Additionally, neurological symptoms, including learning and memory processes, may be related to the high or toxic concentrations of the sweetener metabolites.

  5. Visible-light-activated photoelectrochemical biosensor for the study of acetylcholinesterase inhibition induced by endogenous neurotoxins.

    PubMed

    Huang, Qilin; Chen, Hua; Xu, Lili; Lu, Danqin; Tang, Linlin; Jin, Litong; Xu, Zhiai; Zhang, Wen

    2013-07-15

    In this report, a novel visible-light-activated photoelectrochemical biosensor was fabricated to study the inhibition of acetylcholinesterase (AChE) activity induced by two endogenous neurotoxins, 1(R)-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-Sal] and 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetra-hydroisoquinoline [(R)-NMSal], which have drawn much attention in the study of the pathogenesis of neurodegenerative diseases such as Parkinson's disease. The photoelectrode was prepared by three steps, as follows. At first, nitrogen and fluorine co-doped TiO2 nanotubes (TNs) were obtained by anodic oxidation of a Ti sheet. Secondly, silver nanoparticles (AgNPs) were deposited onto the TNs through a microwave-assisted heating polyol (MAHP) process. At last, AChE was immobilized on the obtained photoelectrode and the biosensor was marked as AChE/Ag/NFTNs. Due to the nitrogen and fluorine co-doping, the photoelectrochemical biosensors can produce high photocurrent under visible light irradiation. Moreover, the presence of AgNPs greatly increased the photocurrent response of the biosensor. AChE/Ag/NFTNs hybrid system was used to study AChE inhibition induced by (R)-Sal and (R)-NMSal. The result proved that both (R)-Sal and (R)-NMSal exhibited mixed and reversible inhibition against AChE. This strategy is of great significance for the development of novel photoelectrochemical biosensors in the future.

  6. Design, synthesis and biological evaluation of bambuterol analogues as novel inhibitors of butyrylcholinesterase.

    PubMed

    Wu, Jie; Tian, Yiguang; Wang, Shanping; Pistolozzi, Marco; Jin, Ya; Zhou, Ting; Roy, Gaurab; Xu, Ling; Tan, Wen

    2017-01-27

    An increase activity of butyrylcholinesterase is believed to contribute to Alzheimer's disease. Bambuterol is a known potent inhibitor of butyrylcholinesterase, but it has undesired cardiac effects and less lipophilicity. Thirteen bambuterol analogues were synthesized using 1-(3, 5-dihydroxyphenyl) ethanone as a starting material. In-vitro cholinesterase assay established that the majority of the compounds are specific butyrylcholinesterase inhibitors. Out of the 13 compounds, two bambuterol derivatives, BD-6 and BD-11 exhibited similar efficacies in inhibiting butyrylcholinesterase with fewer effects on heart and enhanced possibilities of permeating through the blood-brain barrier as compared to bambuterol. These bambuterol analogues may provide better alternatives for treatments of Alzheimer's disease.

  7. Application of a dynamic in vitro model with real-time determination of acetylcholinesterase activity for the investigation of tabun analogues and oximes.

    PubMed

    Worek, Franz; Herkert, Nadja M; Koller, Marianne; Thiermann, Horst; Wille, Timo

    2015-12-25

    Tabun-inhibited acetylcholinesterase (AChE) is rather resistant towards reactivation by oximes in vitro while in vivo experiments showed some protection of animals poisoned by this chemical warfare nerve agent after treatment with an oxime and atropine. In addition, AChE inhibited by close tabun analogues, N,N-diethyltabun and N,N-di-n-propyltabun was completely resistant towards reactivation by oximes. In order to get more insight into potential mechanisms of this oxime resistance experiments with these toxic agents and the oximes obidoxime, 2-PAM, MMB-4 and HI-6 were performed utilizing a dynamic model with real-time determination of AChE activity. This experimental setup allowed the investigation of reactivation with minimized side reactions. The determined reactivation constants with tabun-inhibited human AChE were in good agreement with previously reported constants determined with a static model. N,N-diethyl- and N,N-di-n-propyltabun-inhibited human AChE could not be reactivated by oximes which indicates that the inadequate oxime effect was not due to re-inhibition by phosphonyloximes. Additional experiments with tabun-inhibited human and Rhesus monkey AChE revealed that no reactivation occurred with HI-6. These data give further support to the assumption that an interaction of tabun with residues in the active site gorge of AChE prevents effective reactivation by oximes, a mechanism which may also be the reason for the total oxime resistance of N,N-diethyl- and N,N-di-n-propyltabun-inhibited human AChE.

  8. Differential mRNA expression of acetylcholinesterase in the central nervous system of rats with acute and chronic exposure of sarin & physostigmine.

    PubMed

    Bansal, Iti; Waghmare, C K; Anand, T; Gupta, A K; Bhattacharya, B K

    2009-07-01

    A time-course study was carried out to measure the acetylcholinesterase (AChE) gene expression in the brain of female rats exposed to different doses of sarin and physostigmine. Short-term effects were studied with an acute single subcutaneous dose (s.c.) of 80 microg kg(-1) (0.5 x LD(50)) sarin. Cortex and cerebellum showed a significant decline in AChE mRNA expression at 2.5, 24 and 72 h. Biochemical studies showed that plasma butrylcholinesterase (BChE) and brain AChE activities were significantly decreased at 2.5 h, which came back to near control values by 24 h in both cases. For long-term chronic studies, three groups of female rats received daily doses of physostigmine (0.1 mg kg(-1) day(-1)) intramuscularly (i.m.), sarin (15 microg kg(-1) day(-1)) s.c. independently and a combined dose of physostigmine (i.m.) (0.1 mg kg(-1) day(-1)) followed by sarin (s.c.) (15 microg kg(-1) day(-1)) continuously for 30 days. Differential AChE mRNA levels in cortex and cerebellum of rat brain were observed after 30 days and after a lag period of another 30 days with no further administration. Plasma (BChE) and brain (AChE) showed irregular inhibition profile in biochemical studies at 30 days and returned to control levels after 60 days. The acute single subcutaneous administration of sarin for short-term as well as chronic long-term studies showed that AChE inhibition alone does not lead to observed changes in mRNA expression of AChE gene. These observations further suggest that route of administration as well as dose exposure regimen also contributes to the regulation of AChE mRNA expression.

  9. The Proline Rich Tetramerization Peptides in Equine Serum Butyrylcholinesterase

    PubMed Central

    Biberoglu, Kevser; Schopfer, Lawrence M.; Tacal, Ozden; Lockridge, Oksana

    2012-01-01

    Summary Soluble, tetrameric, plasma butyrylcholinesterase from horse has previously been shown to include a non-covalently attached polyproline peptide in its structure. The polyproline peptide matched the polyproline rich region of human lamellipodin. Our goal was to examine the tetramer organizing peptides of horse butyrylcholinesterase in more detail. Horse butyrylcholinesterase was denatured by boiling, thus releasing a set of polyproline peptides ranging in mass from 1173 to 2098 Da. The peptide sequences were determined by fragmentation in the MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometers. Twenty-seven polyproline peptides grouped into 13 families were identified. Peptides contained a minimum of 11 consecutive proline residues and as many as 21. Many of the peptides had a non-proline amino acid at the N-terminus. A search of the protein databanks matched peptides to 9 proteins, though not all peptides matched a known protein. It is concluded that polyproline peptides of various lengths and sequences are included in the tetramer structure of horse BChE. The function of these polyproline peptides is to serve as tetramer organizing peptides. PMID:22889087

  10. Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity.

    PubMed

    Vaughan, A; Rocheleau, T; ffrench-Constant, R

    1997-11-01

    Insecticide resistance is a serious problem facing the effective control of insect vectors of disease. Insensitive acetylcholinesterase (AChE) confers resistance to organophosphorus (OP) and carbamate insecticides and is a widespread resistance mechanism in vector mosquitoes. Although the point mutations that underlie AChE insensitivity have been described from Drosophila, the Colorado potato beetle, and house flies, no resistance associated mutations have been documented from mosquitoes to date. We are therefore using a cloned acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti as a model in which to perform site directed mutagenesis in order to understand the effects of potential resistance associated mutations. The same resistance associated amino-acid replacements as found in other insects also confer OP and carbamate resistance to the mosquito enzyme. Here we describe the levels of resistance conferred by different combinations of these mutations and the effects of these mutations on the kinetics of the AChE enzyme. Over-expression of these constructs in baculovirus will facilitate purification of each of the mutant enzymes and a more detailed analysis of their associated inhibition kinetics.

  11. RNAi of ace1 and ace2 in Blattella germanica reveals their differential contribution to acetylcholinesterase activity and sensitivity to insecticides.

    PubMed

    Revuelta, L; Piulachs, M D; Bellés, X; Castañera, P; Ortego, F; Díaz-Ruíz, J R; Hernández-Crespo, P; Tenllado, F

    2009-12-01

    Cyclorrhapha insect genomes contain a single acetylcholinesterase (AChE) gene while other insects contain at least two ace genes (ace1 and ace2). In this study we tested the hypothesis that the two ace paralogous from Blattella germanica have different contributions to AChE activity, using RNA interference (RNAi) to knockdown each one individually. Paralogous-specific depletion of Bgace transcripts was evident in ganglia of injected cockroaches, although the effects at the protein level were less pronounced. Using spectrophotometric and zymogram measurements, we obtained evidence that BgAChE1 represents 65-75% of the total AChE activity in nerve tissue demonstrating that ace1 encodes a predominant AChE. A significant increase in sensitivity of Bgace1-interfered cockroaches was observed after 48 h of exposure to chlorpyrifos. In contrast, Bgace2 knockdown had a negligible effect on mortality to this organophosphate. These results point out a key role, qualitative and/or quantitative, of AChE1 as target of organophosphate insecticides in this species. Silencing the expression of Bgace1 but not Bgace2 also produced an increased mortality in insects when synergized with lambda-cyhalothrin, a situation which resembles the synergistic effects observed between organophosphates and pyrethroids. Gene silencing of ace genes by RNAi offers an exciting approach for examining a possible functional differentiation in ace paralogous.

  12. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma

    PubMed Central

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Krejci, Eric; Blake, Thomas A.; Johnson, Rudolph C.; Masson, Patrick; Lockridge, Oksana

    2016-01-01

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immunoextract BChE from the plasma of humans, monkey and other animals. PMID:26585590

  13. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma.

    PubMed

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Krejci, Eric; Blake, Thomas A; Johnson, Rudolph C; Masson, Patrick; Lockridge, Oksana

    2016-01-05

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.

  14. Benzophenone-based derivatives: a novel series of potent and selective dual inhibitors of acetylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation.

    PubMed

    Belluti, Federica; Bartolini, Manuela; Bottegoni, Giovanni; Bisi, Alessandra; Cavalli, Andrea; Andrisano, Vincenza; Rampa, Angela

    2011-05-01

    The leading mechanistic theory of Alzheimer's disease (AD) is the "amyloid hypothesis" which states that the accumulation of the amyloid β protein (Aβ), and its subsequent aggregation into plaques, is responsible for the initiation of a cascade of events resulting in neurodegeneration and dementia. The anti-amyloid disease-modifying approach, based on the decrease in the production of Aβ, gained thus a paramount importance. The aim of this study was the design and synthesis of a new series of acetylcholinesterase inhibitors (AChEIs) endowed with anti-Aβ aggregating capability. These dual binding inhibitors, being able to interact both with the peripheral anionic site (PAS) of AChE and the catalytic subsite, proved to be able to inhibit the AChE-induced Aβ aggregation. Thus, starting from the lead compound 1, an AChEI composed by a benzophenone scaffold and a N,N'-methylbenzylamino group, a substantial modification aimed at targeting the PAS was performed. To this aim, different amino-terminal side chains were incorporated into this main framework, in order to mimic the diethylmethylammonium alkyl moiety of the pure PAS ligand propidium. The synthesized compounds proved to effectively and selectively inhibit AChE. Moreover, compounds 16a-c and 18a,b, with a propoxy and a hexyloxy tether respectively, showed a good activity against the AChE-induced Aβ aggregation. In particular, molecular modeling studies confirmed that compounds carrying the diethylaminopropoxy and the diethylaminohexyloxy side chains (compounds 16a and 19a, respectively) could suitably contact the PAS pocket of the enzyme.

  15. Pyridoxine-resveratrol hybrids Mannich base derivatives as novel dual inhibitors of AChE and MAO-B with antioxidant and metal-chelating properties for the treatment of Alzheimer's disease.

    PubMed

    Yang, Xia; Qiang, Xiaoming; Li, Yan; Luo, Li; Xu, Rui; Zheng, Yunxiaozhu; Cao, Zhongcheng; Tan, Zhenghuai; Deng, Yong

    2017-04-01

    A series of pyridoxine-resveratrol hybrids Mannich base derivatives as multifunctional agents have been designed, synthesized and evaluated for cholinesterase (ChE) and monoamine oxidase (MAO) inhibitory activity. To further explore the multifunctional properties of the new derivatives, their antioxidant activities and metal-chelating properties were also tested. The results showed that most of these compounds could selectively inhibit acetylcholinesterase (AChE) and MAO-B. Among them, compounds 7d and 8b exhibited the highest potency for AChE inhibition with IC50 values of 2.11μM and 1.56μM, respectively, and compound 7e exhibited the highest MAO-B inhibition with an IC50 value of 2.68μM. The inhibition kinetic analysis revealed that compound 7d showed a mixed-type inhibition, binding simultaneously to the CAS and PAS of AChE. Molecular modeling study was also performed to investigate the binding mode of these hybrids with MAO-B. In addition, all target compounds displayed good antioxidant and metal-chelating properties. Taken together, these preliminary findings can be a new starting point for further development of multifunctional agents for Alzheimer's disease.

  16. Identification of Potential Herbal Inhibitor of Acetylcholinesterase Associated Alzheimer's Disorders Using Molecular Docking and Molecular Dynamics Simulation

    PubMed Central

    Seniya, Chandrabhan; Khan, Ghulam Jilani; Uchadia, Kuldeep

    2014-01-01

    Cholinesterase inhibitors (ChE-Is) are the standard for the therapy of AD associated disorders and are the only class of approved drugs by the Food and Drug Administration (FDA). Additionally, acetylcholinesterase (AChE) is the target for many Alzheimer's dementia drugs which block the function of AChE but have some side effects. Therefore, in this paper, an attempt was made to elucidate cholinesterase inhibition potential of secondary metabolite from Cannabis plant which has negligible or no side effect. Molecular docking of 500 herbal compounds, against AChE, was performed using Autodock 4.2 as per the standard protocols. Molecular dynamics simulations have also been carried out to check stability of binding complex in water for 1000 ps. Our molecular docking and simulation have predicted high binding affinity of secondary metabolite (C28H34N2O6) to AChE. Further, molecular dynamics simulations for 1000 ps suggest that ligand interaction with the residues Asp72, Tyr70-121-334, and Phe288 of AChE, all of which fall under active site/subsite or binding pocket, might be critical for the inhibitory activity of AChE. This approach might be helpful to understand the selectivity of the given drug molecule in the treatment of Alzheimer's disease. The study provides evidence for consideration of C28H34N2O6 as a valuable small ligand molecule in treatment and prevention of AD associated disorders and further in vitro and in vivo investigations may prove its therapeutic potential. PMID:25054066

  17. Identification of potential herbal inhibitor of acetylcholinesterase associated Alzheimer's disorders using molecular docking and molecular dynamics simulation.

    PubMed

    Seniya, Chandrabhan; Khan, Ghulam Jilani; Uchadia, Kuldeep

    2014-01-01

    Cholinesterase inhibitors (ChE-Is) are the standard for the therapy of AD associated disorders and are the only class of approved drugs by the Food and Drug Administration (FDA). Additionally, acetylcholinesterase (AChE) is the target for many Alzheimer's dementia drugs which block the function of AChE but have some side effects. Therefore, in this paper, an attempt was made to elucidate cholinesterase inhibition potential of secondary metabolite from Cannabis plant which has negligible or no side effect. Molecular docking of 500 herbal compounds, against AChE, was performed using Autodock 4.2 as per the standard protocols. Molecular dynamics simulations have also been carried out to check stability of binding complex in water for 1000 ps. Our molecular docking and simulation have predicted high binding affinity of secondary metabolite (C28H34N2O6) to AChE. Further, molecular dynamics simulations for 1000 ps suggest that ligand interaction with the residues Asp72, Tyr70-121-334, and Phe288 of AChE, all of which fall under active site/subsite or binding pocket, might be critical for the inhibitory activity of AChE. This approach might be helpful to understand the selectivity of the given drug molecule in the treatment of Alzheimer's disease. The study provides evidence for consideration of C28H34N2O6 as a valuable small ligand molecule in treatment and prevention of AD associated disorders and further in vitro and in vivo investigations may prove its therapeutic potential.

  18. Effects of intralipid and caffeic acid phenethyl ester on neurotoxicity, oxidative stress, and acetylcholinesterase activity in acute chlorpyriphos intoxication

    PubMed Central

    Ozkan, Umit; Osun, Arif; Basarslan, Kagan; Senol, Serkan; Kaplan, Ibrahim; Alp, Harun

    2014-01-01

    Chlorpyriphos is one of the most widely used organophosphate (OP) insecticide in agriculture with potential toxicity. Current post-exposure treatments consist of anti-cholinergic drugs and oxime compounds. We studied the effects of intralipid and caffeic acid phenethyl ester (CAPE) on chlorpyriphos toxicity to compose an alternative or supportive treatment for OP poisoning. Methods: Forty-nine rats were randomly divided into seven groups. Chlorpyriphos was administered for toxicity. Intralipid (IL) and CAPE administered immediately after chlorpyriphos. Serum acetylcholinesterase (AChE) level, total oxidant status (TOS), total antioxidant response (TAR), and histologic examination of cerebellum and brain tissue with Hematoxylin-Eosin and immunohistochemical dyes were examined. Results: Serum enzym levels showed that chlorpyriphos and CAPE inhibited AChE while IL alone had no effect, chlorpyriphos and CAPE intensifies the inhibition effect. Significant difference at AChE levels between the chlorpyriphos+IL and chlorpyriphos+CAPE verified that IL has a protective effect on AChE inhibition. TAR levels were significantly increased in all groups except chlorpyriphos group, TOS levels revealed that CAPE and IL decrease the amount of oxidative stress. Histologic examination revealed that neuronal degeneration was slightly decreased at chlorpyriphos+IL group, but CAPE had a significant effect on protection of neuronal degeneration. Conclusion: The results of this study gave us three key points. 1) AChE activity is important for diagnosis of OP intoxication but it has no value for determining the neuro-degeneration. 2) CAPE inhibits AChE activity and may increase the muscarinic-nicotinic hyperactivation. Therefore it should not be used for treatment of OP intoxication. 3) IL decreases the severity of neurodegeneration and symptoms of OP intoxication and it can be used as a supportive agent. PMID:24955152

  19. Comparison of Chlorpyrifos-Oxon and Paraoxon Acetylcholinesterase Inhibition Dynamics: Potential role of a peripheral binding site

    SciTech Connect

    Kousba, Ahmed A.; Sultatos, L G.; Poet, Torka S.; Timchalk, Chuck

    2004-08-02

    The primary mechanism of action for organophosphorus (OP) insecticides involves the inhibition of acetylcholinesterase (AChE) by oxygenated metabolites (oxons). This inhibition has been attributed to the phosphorylation of the serine hydroxyl group located in the active site of the AChE molecule. The rate of phosphorylation is described by the bimolecular inhibitory rate constant (ki), which has been utilized for quantification of OP inhibitory capacity. It has been previously proposed that a peripheral binding site exists on the AChE molecule, which when occupied, reduces the capacity of additional oxon molecules to phosphorylate the active site. The objective of the current study was to evaluate the interaction of chlorpyrifos oxon (CPO) and paraoxon (PO) with rat brain AChE using a modified Ellman assay in conjunction with a pharmacodynamic model to further assess the dynamics of AChE inhibition and the potential role of a peripheral binding site. The ki for AChE inhibition determined at oxon concentrations of 5 x 10{sup -4} 100 nM were 0.212 and 0.0216 nM-1h-1 for CPO and PO, respectively. The spontaneous reactivation rates of the inhibited AChE for CPO and PO were 0.087 and 0.078 h-1, respectively. In contrast, the ki estimated at a low oxon concentration (1 pM) were {approx} 1,000 and 10,000 -fold higher than those determined at high CPO and PO concentrations, respectively. At these low concentrations, the ki estimates were approximately similar for both CPO and PO (180 and 250 nM-1h-1, respectively). This implies that at low exposure concentrations, both oxons exhibited similar inhibitory potency in contrast to the marked difference exhibited at higher concentrations, which is consistent with the presence of a peripheral binding site on the AChE enzyme. These results support the potential importance of a secondary binding site associated with AChE kinetics, particularly at low environmentally relevant concentrations.

  20. Architectonic subdivisions of the motor thalamus of owl monkeys: Nissl, acetylcholinesterase, and cytochrome oxidase patterns.

    PubMed

    Stepniewska, I; Preuss, T M; Kaas, J H

    1994-11-22

    As the first part of an investigation of the motor thalamus and its cortical connections in the owl monkey, a New World anthropoid primate, we studied thalamic architecture by using stains for Nissl, acetylcholinesterase (AChE), and cytochrome oxidase (CO), in order to identify subdivisions of the ventrolateral thalamic region as well as other nuclei with motor connections. Material was obtained from brains cut in the frontal, horizontal, and parasagittal planes. Our results indicate that the ventrolateral thalamic region (VL) of owl monkeys is a heterogeneous structure composed of several architectonic subdivisions that resemble divisions that have been described in macaques and other Old World anthropoids. All of these subdivisions are more readily distinguished in AChE than in Nissl or CO preparations. The anterior part of VL, VLa (VLo of Olszewski), is characterized by clusters of medium-sized, darkly stained neurons. VLa is also distinguished by AChE-positive cells embedded in a matrix of neurites as well as by a characteristic dark, irregular net of blood vessels. The posterior part of VL is rather uniform cytoarchitectonically and contains large, darkly stained, and sparsely distributed neurons. However, we were able to distinguish three subdivisions of posterior VL that closely correspond to structures described by Olszewski in macaques: a principal segment, VLp (VPLo of Olszewski), a medial segment, VLx ("area X" of Olszewski), and a dorsal segment, VLd (VLc and VLps of Olszewski). In AChE, VLd is much darker than the other divisions. The distinction between VLp and VLx, which together make up the largest part of VL, is less marked, although VLp is somewhat darker and more irregular in appearance in AChE than is VLx.

  1. Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

    PubMed Central

    Marchot, P.; Ravelli, R. B.; Raves, M. L.; Bourne, Y.; Vellom, D. C.; Kanter, J.; Camp, S.; Sussman, J. L.; Taylor, P.

    1996-01-01

    A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions. PMID:8845756

  2. LWH and ACH Helmet Hardware Study

    DTIC Science & Technology

    2015-11-30

    screws and nuts used with the Light Weight Helmet (LWH) and Advanced Combat Helmet (ACH). The testing included basic dimensional measurements, Rockwell...laboratory tests to characterize the properties of helmet screws and nuts used with the Light Weight Helmet (LWH) and Advanced Combat Helmet (ACH). The

  3. Synthesis and in silico evaluation of 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives against Alzheimer disease: to understand their interacting mechanism with acetylcholinesterase.

    PubMed

    Kannan, M; Manivel, P; Geetha, K; Muthukumaran, J; Rao, H Surya Prakash; Krishna, R

    2012-01-01

    Anomalous action of human acetylcholinesterase (hAChE) in Alzheimer's disease (AD) was restrained by various AChE inhibitors, of which the specific and potent lead candidate Donepezil is used for treating the disease AD. Besides the specificity, the observed undesirable side effects caused by Donepezil invoked the quest for new lead molecules with the increased potency and specificity for AChE. The present study elucidates the potency of six 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives to form a specific interaction with the peripheral anionic site and catalytic anionic subsite residues of hAChE. The NMSMs were prepared in good yield from 1,1-di(methylsulfanyl)-2-nitroethylene and primary amine (or) amino acid esters. In silico interaction analysis reveals specific and potent interactions between hAChE and selected ligand molecules. The site-specific interactions formed between these molecules also results in a conformational change in the orientation of active site residues of hAChE, which prevents them from being accessed by beta-amyloid protein (Aβ), which is a causative agent for amyloid plaque formation and acetylcholine (ACh). In silico interaction analysis between the ligand-bounded hAChE with Aß and ACh confirms this observation. The variation in the conformation of hAChE associated with the decreased ability of Aβ and ACh to access the respective functional residues of hAChE induced by the novel NMSMs favors their selection for in vivo analysis to present themselves as new members of hAChE inhibitors.

  4. Nanoparticle-Based Electrochemical Immunosensor for the Detection of Phosphorylated Acetylcholinesterase: An Exposure Biomarker of Organophosphate Pesticides and Nerve AgentsOrganophosphate Pesticides and Nerve Agents

    SciTech Connect

    Liu, Guodong; Wang, Jun; Barry, Richard C.; Petersen, Catherine E.; Timchalk, Charles; Gassman, Paul L.; Lin, Yuehe

    2008-11-01

    A nanoparticle-based electrochemical immunosensor has been developed for the detection of phosphorylated acetylcholinesterase (AChE) adducts, which is a potential exposure biomarker for organophosphate pesticides (OP) and chemical warfare nerve agent exposures. Zirconia nanoparticles (ZrO2 NPs) were used as selective sorbents to capture the phosphorylated AChE adduct, and quantum dots (ZnS@CdS, QDs) were used as tags to label monoclonal anti-AChE antibody to track the immunorecognition events. The sandwich-like immunoreactions were performed among the ZrO2 NPs, which were pre-coated on a screen printed electrode (SPE) by electrodeposition, phosphorylated AChE and QD-anti-AChE. The captured QD tags were determined on the SPE by electrochemical stripping analysis of its metallic component (cadmium) after an acid-dissolution step. Paraoxon was used as a model OP insecticide to prepare the phosphorylated AChE adduct to demonstrate the proof of principle for this sensor technology. The paraoxon-AChE adduct was characterized by Fourier Transform Infrared Spectrum, and the binding affinity of anti-AChE to the paraoxon-AChE was validated with an enzyme-linked immunosorbent assay. The parameters (e.g., amount of ZrO2 NP, QD-anti-AChE concentration,) that govern the electrochemical response of immunosensors were optimized. The voltammetric response of the immunosensor is highly linear over the range of 10 pM to 4 nM paraoxon-AChE, and the limit of detection is estimated to be 8 pM. This new nanoparticle-based electrochemical immunosensor thus provides a sensitive and quantitative tool for biomonitoring exposure to OP pesticides and nerve agents.

  5. Comparative investigation between acetylcholinesterase obtained from commercial sources and genetically modified Drosophila melanogaster: application in amperometric biosensors for methamidophos pesticide detection.

    PubMed

    de Oliveira Marques, Paulo Roberto Brasil; Nunes, Gilvanda Silva; dos Santos, Teresa Cristina Rodrigues; Andreescu, Silvana; Marty, Jean-Louis

    2004-11-01

    Genetically modified acetylcholinesterase (AChE) from Drosophila melanogaster (dm) and from commercial sources, Electric eel (ee), Bovine erythrocites (be) and Human erythrocites (he), were investigated as biological receptors for the detection of methamidophos pesticide based on inhibition studies. Most engineered variant of AChE from dm showed enhanced sensitivity toward methamidophos pesticide. Among 24 dmAChE variants tested, 12 presented a sensitivity comparable to the commercially available eeAChE, but higher than AChEs from be and he. Four were found more sensitive and six others were insensitive to methamidophos insecticide. The D375G,Y370F,Y374A,F376L mutant was the most sensitive, with a ki value of 2.2 X 10(6) mol(-1) L min(-1), three orders of magnitude higher than eeAChE (1.1 X 10(3) mol(-1) L min(-1)). The sensor constructed with genetically modified enzyme showed better characteristics with respect to detection limit and sensitivity compared with those using commercial eeAChE. Differential pulse polarography and chronoamperometry were used as electrochemical techniques to characterize the AChE biosensors. The lower detection limit of 1 ppb was obtained with D375G,Y370F,Y374A,F376L mutant of dmAChE, compared to 90 ppb for the commercial eeAChE. This study may stimulate scientists to develop more sensitive and selective procedures for organophosphorus insecticides detection by using engineered variant of dmAChE.

  6. Increased Acetylcholinesterase Expression in Bumble Bees During Neonicotinoid-Coated Corn Sowing.

    PubMed

    Samson-Robert, Olivier; Labrie, Geneviève; Mercier, Pierre-Luc; Chagnon, Madeleine; Derome, Nicolas; Fournier, Valérie

    2015-07-30

    While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChE) activity in honey bees at sublethal doses. High AChE levels may therefore act as a biomarker of exposure to neonicotinoids. This two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥ 3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoid-treated seeds), and maintained for the duration of corn sowing. As determined by Real Time qPCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication.

  7. Bioactive Paper Sensor Based on the Acetylcholinesterase for the Rapid Detection of Organophosphate and Carbamate Pesticides

    PubMed Central

    Badawy, Mohamed E. I.; El-Aswad, Ahmed F.

    2014-01-01

    In many countries, people are becoming more concerned about pesticide residues which are present in or on food and feed products. For this reason, several methods have been developed to monitor the pesticide residue levels in food samples. In this study, a bioactive paper-based sensor was developed for detection of acetylcholinesterase (AChE) inhibitors including organophosphate and carbamate pesticides. Based on the Ellman colorimetric assay, the assay strip is composed of a paper support (1 × 10 cm), onto which a biopolymer chitosan gel immobilized in crosslinking by glutaraldehyde with AChE and 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) and uses acetylthiocholine iodide (ATChI) as an outside reagent. The assay protocol involves introducing the sample to sensing zone via dipping of a pesticide-containing solution. Following an incubation period, the paper is placed into ATChI solution to initiate enzyme catalyzed hydrolysis of the substrate, causing a yellow color change. The absence or decrease of the yellow color indicates the levels of the AChE inhibitors. The biosensor is able to detect organophosphate and carbamate pesticides with good detection limits (methomyl = 6.16 × 10−4 mM and profenofos = 0.27 mM) and rapid response times (~5 min). The results show that the paper-based biosensor is rapid, sensitive, inexpensive, portable, disposable, and easy-to-use. PMID:25484901

  8. Acetylcholinesterase Inhibitors Assay Using Colorimetric pH Sensitive Strips and Image Analysis by a Smartphone

    PubMed Central

    Kostelnik, Adam; Cegan, Alexander

    2017-01-01

    Smartphones are widely spread and their usage does not require any trained personnel. Recently, smartphones were successfully used in analytical chemistry as a simple detection tool in some applications. This paper focuses on immobilization of acetylcholinesterase (AChE) onto commercially available pH strips with stabilization in the gelatin membrane. AChE degrades acetylcholine into choline and acetic acid which causes color change of acid-base indicator. Smartphone served as a tool for measurement of indicator color change from red to orange while inhibitors blocked this process. AChE inhibitors were measured with limits of detection, 149 nM and 22.3 nM for galanthamine and donepezil, respectively. Organic solvents were measured for method interferences. Measurement procedure was performed on 3D printed holder and digital photography was evaluated using red-green-blue (RGB) channels. The invented assay was validated to the standard Ellman's test and verified on murine plasma samples spiked with inhibitors. We consider that the assay is fully suitable for practical performance. PMID:28286520

  9. Protective Effect of Thunbergia laurifolia (Linn.) on Lead Induced Acetylcholinesterase Dysfunction and Cognitive Impairment in Mice

    PubMed Central

    Phyu, Moe Pwint; Tangpong, Jitbanjong

    2013-01-01

    Thunbergia laurifolia (linn., TL), a natural phenolic compound, has been reported to have many benefits and medicinal properties. The current study ascertains the total phenolic content present in TL aqueous leaf extract and also examines the antioxidant ability of the extract in preserving acetylcholinesterase (AChE) activity of mice exposed to lead in vivo and in vitro model. Mice were given lead acetate (Pb) in drinking water (1 g/L) together with TL 100 and 200 mg/kg/day. The result showed that Pb induced AChE dysfunction in both in vitro and in vivo studies. TL significantly prevented Pb induced neurotoxicity in a dose-dependent manner which was indicated by comparatively better performance of TL treated mice in Morris Water Maze Swimming Test and increased AChE activity in the tissue sample collected from the brains of these mice. TL also exhibited the greatest amount of phenolic content, which has a significant positive correlation with its antioxidant capacity (P < 0.05). Taken together, these data suggested that the total phenolic compounds in TL could exhibit antioxidant and in part neuroprotective properties. It may play a potential treatment strategy for Pb contamination. PMID:24455676

  10. Mouse Acetylcholinesterase Unliganded and in Complex with Huperzine A: A Comparison of Molecular Dynamics Simulations

    SciTech Connect

    Tara, Sylvia; Straatsma, TP; Mccammon, Andy

    1999-06-01

    A 1 ns molecular dynamics simulation of unliganded mouse acetylcholinesterase (AChE) is compared to a previous simulation of mouse AChE complexed with Huperzine A (HupA). Several common features are observed. In both simulations, the active site gorge fluctuates in size during the 1 ns trajectory, and is completely pinched off several times. Many of the residues in the gorge that formed hydrogen bonds with HupA in the simulation of the complex, now form hydrogen bonds with other protein residues and water molecules in the gorge. The opening of a "backdoor" entrance to the active site that was found in the simulation of the complex is also observed in the unliganded simulation. Differences between the two simulations include overall lower structural RMS deviations for residues in the gorge in the unliganded simulation, a smaller diameter of the gorge in the absence of HupA, and the disappearance of a side channel that was frequently present in the liganded simulation. The differences between the two simulations can be attributed, in part, to the interaction of AChE with HupA.

  11. Acetylcholinesterase based biosensor for monitoring of Malathion and Acephate in food samples: a voltammetric study.

    PubMed

    Raghu, P; Madhusudana Reddy, T; Reddaiah, K; Kumara Swamy, B E; Sreedhar, M

    2014-01-01

    Acetylcholinesterase (AChE) biosensor was developed through silica sol-gel (SiSG) immobilisation of AChE on the carbon paste electrode (CPE) and used as working electrode. AChE catalyses the cleavage of acetylthiocholine chloride (ASChCl or substrate) to thiocholine, which was oxidised to give a disulphide compound by dimerisation at 0.60V versus saturated calomel electrode. All the experiments were carried out in 0.1M phosphate buffer solution (PBS) at pH 7.0 and 0.1M KCl solution at room temperature. The limit of detection and limit of quantification values were found to be 0.058ppm, 0.044ppm and 0.194ppm, 0.147ppm for Malathion and Acephate, respectively. The response of the biosensor showed a good linearity range with an incubation time of 4min for Malathion and Acephate, respectively. This biosensor was used for the direct determination of pesticides without any pretreatment and it requires less time for analysis.

  12. Effect of glyphosate-based herbicide on acetylcholinesterase activity in tadpoles, Hoplobatrachus rugulosus.

    PubMed

    Ruamthum, W; Visetson, S; Milne, J R; Bullangpoti, V

    2011-01-01

    This study focused on the effects of a glyphosate-based herbicide on activity of the neuron enzyme, acetylcholinesterase (AchE), in the tadpole stage (stage 35-39) of the East Asian Bullfrog, Hoplobatrachus rugulosus. There were 4 herbicide concentration treatments consisting of glyphosate-based herbicide added at 21, 24, 27 and 30 microl to 1L de-chlorinated water in glass containers (10x15x20 cm). There were 4 replicates per treatment, each replicate using 20 tadpoles. The toxicity results were compared with tadpoles in distilled water as a control treatment. After 24, 48, 72 and 96 hours exposure to glyphosate-based herbicide concentrations, LC50 values of 25.21, 24.66, 24.16 and 23.63 microl/L, respectively, were recorded. AChE activities decreased significantly and markedly with herbicide concentration. Such inhibition of AChE activity by this glyphosate-based herbicide indicates the potential of such herbicides to disrupt ecological communities in water near where the herbicides are applied.

  13. Acetylcholinesterase Inhibitors Assay Using Colorimetric pH Sensitive Strips and Image Analysis by a Smartphone.

    PubMed

    Kostelnik, Adam; Cegan, Alexander; Pohanka, Miroslav

    2017-01-01

    Smartphones are widely spread and their usage does not require any trained personnel. Recently, smartphones were successfully used in analytical chemistry as a simple detection tool in some applications. This paper focuses on immobilization of acetylcholinesterase (AChE) onto commercially available pH strips with stabilization in the gelatin membrane. AChE degrades acetylcholine into choline and acetic acid which causes color change of acid-base indicator. Smartphone served as a tool for measurement of indicator color change from red to orange while inhibitors blocked this process. AChE inhibitors were measured with limits of detection, 149 nM and 22.3 nM for galanthamine and donepezil, respectively. Organic solvents were measured for method interferences. Measurement procedure was performed on 3D printed holder and digital photography was evaluated using red-green-blue (RGB) channels. The invented assay was validated to the standard Ellman's test and verified on murine plasma samples spiked with inhibitors. We consider that the assay is fully suitable for practical performance.

  14. Maternal caffeine exposure alters neuromotor development and hippocampus acetylcholinesterase activity in rat offspring.

    PubMed

    Souza, Ana Claudia; Souza, Andressa; Medeiros, Liciane Fernandes; De Oliveira, Carla; Scarabelot, Vanessa Leal; Da Silva, Rosane Souza; Bogo, Mauricio Reis; Capiotti, Katiucia Marques; Kist, Luiza Wilges; Bonan, Carla D; Caumo, Wolnei; Torres, Iraci L S

    2015-01-21

    The objective of this study was to evaluate the effects of maternal caffeine intake on the neuromotor development of rat offspring and on acetylcholine degradation and acetylcholinesterase (AChE) expression in the hippocampus of 14-day-old infant rats. Rat dams were treated with caffeine (0.3g/L) throughout gestation and lactation until the pups were 14 days old. The pups were divided into three groups: (1) control, (2) caffeine, and (3) washout caffeine. The washout group received a caffeine solution until the seventh postnatal day (P7). Righting reflex (RR) and negative geotaxis (NG) were assessed to evaluate postural parameters as an index of neuromotor reflexes. An open-field (OF) test was conducted to assess locomotor and exploratory activities as well as anxiety-like behaviors. Caffeine treatment increased both RR and NG latency times. In the OF test, the caffeine group had fewer outer crossings and reduced locomotion compared to control, while the washout group showed increased inner crossings in relation to the other groups and fewer rearings only in comparison to the control group. We found decreased AChE activity in the caffeine group compared to the other groups, with no alteration in AChE transcriptional regulation. Chronic maternal exposure to caffeine promotes important alterations in neuromotor development. These results highlight the ability of maternal caffeine intake to interfere with cholinergic neurotransmission during brain development.

  15. Increased Acetylcholinesterase Expression in Bumble Bees During Neonicotinoid-Coated Corn Sowing

    PubMed Central

    Samson-Robert, Olivier; Labrie, Geneviève; Mercier, Pierre-Luc; Chagnon, Madeleine; Derome, Nicolas; Fournier, Valérie

    2015-01-01

    While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChE) activity in honey bees at sublethal doses. High AChE levels may therefore act as a biomarker of exposure to neonicotinoids. This two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoid-treated seeds), and maintained for the duration of corn sowing. As determined by Real Time qPCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication. PMID:26223214

  16. Blocked Enzymatic Etching of Gold Nanorods: Application to Colorimetric Detection of Acetylcholinesterase Activity and Its Inhibitors.

    PubMed

    Saa, Laura; Grinyte, Ruta; Sánchez-Iglesias, Ana; Liz-Marzán, Luis M; Pavlov, Valeri

    2016-05-04

    The anisotropic morphology of gold nanorods (AuNRs) has been shown to lead to nonuniform ligand distribution and preferential etching through their tips. We have recently demonstrated that this effect can be achieved by biocatalytic oxidation with hydrogen peroxide, catalyzed by the enzyme horseradish peroxidase (HRP). We report here that modification of AuNRs with thiol-containing organic molecules such as glutathione and thiocholine hinders enzymatic AuNR etching. Higher concentrations of thiol-containing molecules in the reaction mixture gradually decrease the rate of enzymatic etching, which can be monitored by UV-vis spectroscopy through changes in the AuNR longitudinal plasmon band. This effect can be applied to develop novel optical assays for acetylcholinesterase (AChE) activity. The biocatalytic hydrolysis of acetylthiocholine by AChE yields thiocholine, which prevents enzymatic AuNR etching in the presence of HRP. Additionally, the same bioassay can be used for the detection of nanomolar concentrations of AChE inhibitors such as paraoxon and galanthamine.

  17. The action of two ethyl carbamates on acetylcholinesterase and reproductive organs of Rhipicephalus microplus.

    PubMed

    Prado-Ochoa, M G; Ramírez-Noguera, P; Díaz-Torres, R; Garrido-Fariña, G I; Vázquez-Valadez, V H; Velázquez-Sánchez, A M; Muñoz-Guzmán, M A; Angeles, E; Alba-Hurtado, F

    2014-01-31

    The effects produced by the new synthetic carbamates ethyl-(4-bromophenyl) carbamate and ethyl-(4-chlorophenyl) carbamate on the acetylcholinesterase (AChE) activity, egg structure and reproductive organs of two Rhipicephalus microplus strains were evaluated. Inhibition kinetic parameters showed that the studied carbamates are weak inhibitors and have a low affinity for R. microplus AChE. Histologically, in oocytes from carbamate-treated engorged female ticks, a loss of shape, cytoplasmic vacuoles, decreased chorion deposition, alterations in cytoplasmic granularity and irregular membranes were observed. In oocyte germinal vesicles, a loss of shape, nucleolar fragmentation and membrane alterations with degenerative signs were observed. The ovarian epithelium was vacuolated, flattened, eroded and contained pyknotic nuclei. These alterations were observed from the first day and persisted and increased in severity until day 7 post-treatment. The ovaries from carbamate-treated ticks had fewer stage IV-V oocytes and more stage I-II oocytes. Additionally, eggs produced by the treated ticks had a modified appearance, decreased size, a reduced superficial waxy layer and a loss of viability. The results of this study show that the effects of carbamates on R. microplus were independent of AChE inhibition and show that the morphological alterations in the reproductive organs were due to carbamate actions on the vitellogenesis and viability of the ovarian cells.

  18. A fluorometric assay for acetylcholinesterase activity and inhibitor detection based on DNA-templated copper/silver nanoclusters.

    PubMed

    Li, Wenhua; Li, Wang; Hu, Yufang; Xia, Yalin; Shen, Qinpeng; Nie, Zhou; Huang, Yan; Yao, Shouzhuo

    2013-09-15

    A novel label-free, rapid, cost-effective, and highly sensitive fluorometric sensor has been constructed for the detection of acetylcholinesterase (AChE) activity and its inhibitor based on the fluorescence quenching of DNA-templated copper/silver nanoclusters (DNA-Cu/AgNCs). In this assay, AChE catalyzes the hydrolysis of acetylthiocholine (ATCh) to form thiocholine which induces fluorescence quenching of DNA-Cu/AgNCs. The AChE activity could be detected as low as 0.05mU/mL and with a linear range from 0.05 to 2.0mU/mL. This assay offers a very convenient "mix and detect" approach for AChE activity. On the other hand, tacrine and organophosphorus pesticides (OPPs) were employed to inhibit the hydrolysis of ATCh, which could eliminate the fluorescence quenching of DNA-Cu/AgNCs. The IC50 of tacrine and methamidophos were estimated to be 16.9nM and 0.075mg/L, respectively. This method was also used to detect spiked OPPs in agricultural products successfully. The present work may expand the use of DNA-Cu/AgNCs to the field of enzyme sensors.

  19. Acetylcholinesterases from the Disease Vectors Aedes aegypti and Anopheles gambiae: Functional Characterization and Comparisons with Vertebrate Orthologues

    PubMed Central

    Engdahl, Cecilia; Knutsson, Sofie; Fredriksson, Sten-Åke; Linusson, Anna; Bucht, Göran; Ekström, Fredrik

    2015-01-01

    Mosquitoes of the Anopheles (An.) and Aedes (Ae.) genus are principal vectors of human diseases including malaria, dengue and yellow fever. Insecticide-based vector control is an established and important way of preventing transmission of such infections. Currently used insecticides can efficiently control mosquito populations, but there are growing concerns about emerging resistance, off-target toxicity and their ability to alter ecosystems. A potential target for the development of insecticides with reduced off-target toxicity is the cholinergic enzyme acetylcholinesterase (AChE). Herein, we report cloning, baculoviral expression and functional characterization of the wild-type AChE genes (ace-1) from An. gambiae and Ae. aegypti, including a naturally occurring insecticide-resistant (G119S) mutant of An. gambiae. Using enzymatic digestion and liquid chromatography-tandem mass spectrometry we found that the secreted proteins were post-translationally modified. The Michaelis-Menten constants and turnover numbers of the mosquito enzymes were lower than those of the orthologous AChEs from Mus musculus and Homo sapiens. We also found that the G119S substitution reduced the turnover rate of substrates and the potency of selected covalent inhibitors. Furthermore, non-covalent inhibitors were less sensitive to the G119S substitution and differentiate the mosquito enzymes from corresponding vertebrate enzymes. Our findings indicate that it may be possible to develop selective non-covalent inhibitors that effectively target both the wild-type and insecticide resistant mutants of mosquito AChE. PMID:26447952

  20. Electrochemical detection of malathion pesticide using acetylcholinesterase biosensor based on glassy carbon electrode modified with conducting polymer film.

    PubMed

    Guler, Muhammet; Turkoglu, Vedat; Kivrak, Arif

    2016-06-01

    Acetylcholinesterase (AChE) biosensor based on conducting poly([2,2̍';5̍' 2″]-terthiophene-3̍-carbaldehyde) (PTT) modified glassy carbon electrode (GCE) was constructed. AChE was immobilized on PTT film surface through the covalent bond between aldehyde and amino groups. The properties of PTT modified GCE were studied using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). The biosensor showed an oxidation peak at +0.83 V related to the oxidation of thiocholine, hydrolysis product of acetylthiocholine iodide (ATCI), catalyzed by AChE. The optimum current response of the biosensor was observed at pH 7.5-8.0, 40 °C and 120 U/cm(2) of AChE concentration. The biosensor showed a high sensitivity (183.19 μA/mM), a linear range from 0.015 to 1.644 mM, and a good reproducibility with 1.7 % of relative standard deviation (RSD). The biosensor showed a good stability. The interference of glycin, ascorbic acid, histidine, uric acid, dopamine, and arginine on the biosensor response was studied. An important analytical response from these inteferents that overlaps the biosensor response was not observed. The inhibition rate of malathion as a model pesticide was proportional to its concentrations from 9.99 to 99.01 nM. The detection limit was 4.08 nM.

  1. Effect of a glyphosate-based herbicide in Cyprinus carpio: assessment of acetylcholinesterase activity, hematological responses and serum biochemical parameters.

    PubMed

    Gholami-Seyedkolaei, Seyed Jalil; Mirvaghefi, Alireza; Farahmand, Hamid; Kosari, Ali Asghar

    2013-12-01

    The objective of this study was to investigate the toxicity effects of acute and sublethal of Roundup® as a glyphosate-based herbicide on acetylcholinesterase (AChE) activity and several hematological and biochemical parameters of Cyprinus carpio. The LC₅₀-96 h of Roundup® to C. carpio was found to be 22.19 ppm. Common carp was subjected to Roundup® at 0 (control), 3.5, 7 and 14 ppm for 16 days, and the AChE activity is verified in tissues of gill, muscle, brain and liver. After 5 days, a significant decrease was observed in the AChE activity of muscle, brain and liver tissues. Besides, a time- and dose-dependent increase in mean cell hemoglobin (MCH) and mean cell volume (MCV) was observed. In contrast, a significant decrease was found in the quantities of hemoglobin (Hb), hematocrit (HCT) and, red (RBC) and white (WBC) blood cell count. Also, the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in Roundup® treated groups were significantly higher than the controlled group at experimental periods. However, the level of alkaline phosphatase (ALP) had a significant reduction behavior during the sampling days. It seems that the changes in hematological and biochemical parameters as well as AChE activity could be used as efficient biomarkers in order to determine Roundup® toxicity in aquatic environment.

  2. Kinetics of the postinhibitory reactions of acetylcholinesterase poisoned by chiral isomalathion: a surprising nonreactivation induced by the RP stereoisomers.

    PubMed

    Berkman, C E; Ryu, S; Quinn, D A; Thompson, C M

    1993-01-01

    Inhibitory (ki), spontaneous (k0), and oxime-mediated reactivation (k(oxime)) reaction kinetics for the four stereoisomers of isomalathion (SPRC,SPSC,RPRC, and RPSC) were determined against rat brain acetylcholinesterase (AChE). (SPRC)-Isomalathion was the most potent anticholinesterase agent and RPSC-isomalathion the least potent with racemic material approximately midway in activity. Following inhibition of rat brain AChE by (SPRC)- or (SPSC)-isomalathion, k0 and k(oxime) values were obtained that were comparable to (SP)-isoparathion methyl, indicating that the same mechanism of inhibition was shared, namely, formation of an O,S-dimethyl phosphorothiolated enzyme. Conversely, no appreciable reactivation occurred with or without oxime following inhibition of rat brain AChE by (RPSC)- or (RPRC)-isomalathion. This observation was not consistent with (RP)-isoparathion methyl, and a switch in inhibition mechanism to the loss of the thiomethyl moiety is suggested. The nonreactivation of rat brain AChE following inhibition by the (RP)-isomalathion stereoisomers is postulated to result from a mechanism involving either a beta-elimination of diethyl fumarate or displacement of the thiosuccinate moiety from the phosphate moiety.

  3. Selective and sensitive detection of acetylcholinesterase activity using denatured protein-protected gold nanoclusters as a label-free probe.

    PubMed

    Li, Hongchang; Guo, Yuxin; Xiao, Lehui; Chen, Bo

    2014-01-07

    Based on the fluorescence quenching of novel denatured protein-protected gold nanoclusters, a label-free detection method of acetylcholinesterase (AChE) activity has been developed. Using denatured bovine serum albumin (dBSA), in which 35 cysteine residues can interact polyvalently with Au nanoclusters (AuNCs) as a stabilizing agent, water-soluble and stable fluorescent gold nanoclusters were synthesized. The fluorescence of the AuNCs was quenched by thiocholine that was produced from the AChE hydrolysis of S-acetylthiocholine iodide (ACTI) to detect the AChE activity. The linear range of the method was 0.005-0.15 U mL(-1). The limit of detection (LOD) was 0.02 mU mL(-1). Other enzymes and metal ions, i.e., GPT, γ-GT, GOx, K(+), Ca(2+) and Na(+), showed minimal interference. Using the fluorescence probe, satisfactory results for the detection of the AChE activity in human serum were obtained.

  4. Acetylcholinesterases from the Disease Vectors Aedes aegypti and Anopheles gambiae: Functional Characterization and Comparisons with Vertebrate Orthologues.

    PubMed

    Engdahl, Cecilia; Knutsson, Sofie; Fredriksson, Sten-Åke; Linusson, Anna; Bucht, Göran; Ekström, Fredrik

    2015-01-01

    Mosquitoes of the Anopheles (An.) and Aedes (Ae.) genus are principal vectors of human diseases including malaria, dengue and yellow fever. Insecticide-based vector control is an established and important way of preventing transmission of such infections. Currently used insecticides can efficiently control mosquito populations, but there are growing concerns about emerging resistance, off-target toxicity and their ability to alter ecosystems. A potential target for the development of insecticides with reduced off-target toxicity is the cholinergic enzyme acetylcholinesterase (AChE). Herein, we report cloning, baculoviral expression and functional characterization of the wild-type AChE genes (ace-1) from An. gambiae and Ae. aegypti, including a naturally occurring insecticide-resistant (G119S) mutant of An. gambiae. Using enzymatic digestion and liquid chromatography-tandem mass spectrometry we found that the secreted proteins were post-translationally modified. The Michaelis-Menten constants and turnover numbers of the mosquito enzymes were lower than those of the orthologous AChEs from Mus musculus and Homo sapiens. We also found that the G119S substitution reduced the turnover rate of substrates and the potency of selected covalent inhibitors. Furthermore, non-covalent inhibitors were less sensitive to the G119S substitution and differentiate the mosquito enzymes from corresponding vertebrate enzymes. Our findings indicate that it may be possible to develop selective non-covalent inhibitors that effectively target both the wild-type and insecticide resistant mutants of mosquito AChE.

  5. High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification.

    PubMed

    Richter, Sven; Nieveler, Jens; Schulze, Holger; Bachmann, Till T; Schmid, Rolf D

    2006-04-05

    The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.

  6. THE APPEARANCE OF ACETYLCHOLINESTERASE IN THE MYOTOME OF THE EMBRYONIC RABBIT

    PubMed Central

    Tennyson, Virginia M.; Brzin, Miro; Slotwiner, Paul

    1971-01-01

    Acetylcholinesterase (AChE) activity has been studied in the myoblast of skeletal muscle of the 9–13 day fetal rabbit. Cytochemical activity is present in the nuclear envelope and the endoplasmic reticulum, including its derivatives the subsurface reticulum and the sarcoplasmic reticulum. End product is also found in the Golgi complex of the more differentiated myoblasts. The formation of reticulum-bound acetylcholinesterase in the myoblast appears to be independent of nerve-muscle contact, since the enzyme is present before the outgrowth of the spinal nerve. The nerve lacks cytochemical end product until the myoblast is well differentiated. Possible mechanisms of spontaneous muscle contraction have been discussed. A second type of myotomal cell, which exhibits a poorly localized end product of AChE activity, has been described. The ready solubility of the enzyme or diffusibility of its end product suggests that the enzyme may be a lyoesterase. This cell may be the precursor of the morphologically undifferentiated cell which is closely apposed to the myotubes in later stages of skeletal muscle development. Biochemical studies show a significant increase in AChE activity in the dermomyotome by day 12, when many of the myoblasts are well differentiated and the second type of myotomal cell is prominent. Cytochemical studies have indicated that many of the cells in the sample lack reaction product of enzymic activity, whereas others are very active. Biochemical values, therefore, reflect the amount of enzyme in the dermomyotome as a whole, but give little information on the enzymic content of individual cells. PMID:4256859

  7. Synthesis, Characterization, and Evaluation of Difluoropyrido[4,3-b]indoles as Potential Agents for Acetylcholinesterase and Antiamnesic Activity.

    PubMed

    Madaiah, Malavalli; Jayanna, Bidarur K; Manu, Arakere S; Prashanth, Maralekere K; Revanasiddappa, Hosakere D; Veeresh, Bantal

    2017-04-01

    Acetylcholinesterase (AChE) inhibitors are currently the most widely prescribed drugs for Alzheimer's disease. The high potential of indole compounds in medicinal chemistry led us to discover a novel series of fluoroindole compounds. The synthesis and pharmacological analysis of the difluoropyrido[4,3-b]indoles 11-34 are described. Compounds 11-34 were tested for AChE inhibition activity using a rat brain homogenate. Compounds 25-29 display a promising in vitro profile with an IC50 value range of 46-51.6 nM and show significant protective effect on scopolamine-induced amnesia. The present data indicate that compounds 25-29 may represent attractive potent molecules for the treatment of Alzheimer's disease.

  8. [Distribution of acetylcholinesterase activity in the digestive system of the gastropod molluscs Littorina littorea and Achatina fulica].

    PubMed

    Zaĭtseva, O V; Kuznetsova, T V

    2008-01-01

    With the use of the histochemical procedure for the demonstration of acetylcholinesterase (AchE) activity, the distribution cholinergic regulatory elements was studied in the esophagus, the pharynx, the stomach, the liver (the digestive gland) and the intestine in sea and terrestrial gastropod molluscs that differed in their general organization level, lifestyle, habitat and feeding type. In both molluscs, all the parts of the digestive tract contained the significant amount of intraepithelial AchE-positive cells of the open type, single subepithelial neurons and the nervous fibers localized among the muscle cells of the wall of the organs. The basal processes of the AchE-positive intraepithelial cells were shown to form the intraepithelial nerve plexus and to pass under the epithelium. The peculiarities and common principles in the distribution of the nervous elements detected, their possible function and the regulatory role in the digestion in gastropod molluscs and other animals are discussed.

  9. Acetylcholinesterase inhibition, antioxidant activity and toxicity of Peumus boldus water extracts on HeLa and Caco-2 cell lines.

    PubMed

    Falé, P L; Amaral, F; Amorim Madeira, P J; Sousa Silva, M; Florêncio, M H; Frazão, F N; Serralheiro, M L M

    2012-08-01

    This work aimed to study the inhibition on acetylcholinesterase activity (AChE), the antioxidant activity and the toxicity towards Caco-2 and HeLa cells of aqueous extracts of Peumus Boldus. An IC(50) value of 0.93 mg/mL, for AChE inhibition, and EC(50) of 18.7 μg/mL, for the antioxidant activity, was determined. This activity can be attributed to glycosylated flavonoid derivatives detected, which were the main compounds, although boldine and other aporphine derivatives were also present. No changes in the chemical composition or the biochemical activities were found after gastrointestinal digestion. Toxicity of P. boldus decoction gave an IC(50) value 0.66 mg/mL for HeLa cells, which caused significant changes in the cell proteome profile.

  10. Kinetic characters and resistance to inhibition of crude and purified brain acetylcholinesterase of three freshwater fishes by organophosphates.

    PubMed

    Shaonan, Li; Xianchuan, Xie; Guonian, Zhu; Yajun, Tan

    2004-07-14

    Acetylcholinesterase (AChE) was purified from the brain of three fresh-water fishes, topmouth gudgeon (Pseudorasbora parva), goldfish (Carassius auratus auratus) and rainbow trout (Oncorrhychus mykiss, formerly named Salmo gairdneri) by PEG2000/phosphate-salt two phases extraction, DEAE-Sephadex A-50 and Sephadex G-200 chromatography. Kinetic characters and resistance to inhibition of crude and purified enzymes by organophosphates were then studied. Although the crude enzyme from the trout displayed a different specific activity, kinetic curve, Vmax, and sensitivity to inhibition by oxidized malathion and triazopos compared with the two cyprinoids (i.e. topmouth gudgeon and goldfish), the purified enzymes of all the three species showed no significant difference in all aspects. The result suggested a negligible intrinsic difference of brain AChEs among the tested species.

  11. Acetylcholinesterase activity in Corbicula fluminea Mull., as a biomarker of organophosphate pesticide pollution in Pinacanauan River, Philippines.

    PubMed

    Beltran, Kimberly S; Pocsidio, Glorina N

    2010-06-01

    Organophosphates are known to inhibit the enzyme acetylcholinesterase. In this study, the AChE activity from the total soft tissues of Corbicula fluminea Mull. was used as a biomarker of organophosphate pollution in Pinacanauan River. Clams were collected from two different sites and at different seasons of the year. A colorimetric assay on the total soft tissues of the clams showed a directly proportional relationship between enzyme activity and condition of the riverine system. In vitro experiments on the total soft tissue, adductor muscles, digestive glands, and gills were conducted to assess the degree of localization of AChE as well as the sensitivity and tolerance of the enzymes in these tissues to varying concentrations of malathion. The degree of enzyme localization from highest to lowest is as follows: adductor muscle > gills > digestive gland whereas sensitivity to OP from greatest to least is: gills > adductor muscles > digestive gland.

  12. Is it possible to reverse aged acetylcholinesterase inhibited by organophosphorus compounds? Insight from the theoretical study.

    PubMed

    An, Yun; Zhu, Yali; Yao, Yuan; Liu, Junjun

    2016-04-14

    The main treatment for organophosphorus (OP) compound poisoning in clinics is to restore the activity of acetylcholinesterase (AChE) through oxime-induced reactivation of the phosphorylated OP-AChE adduct. It suffers from a competitive and irreversible aging reaction of the phosphorylated OP-AChE adduct, resulting in permanent inactivity of AChE. However, it was recently reported that N-methyl-2-methoxypyridinium species can act as methylating agents to methylate the methyl methane-phosphonate monoanion, in which the reaction mimics the reverse of the aging reaction of the phosphorylated OP-AChE adduct. If the aging reaction could be really reversed, the efficiency for the OP detoxification should be significantly improved, bringing up the possibility to develop an agent to reverse the aging process of the phosphorylated OP-AChE adduct. However, such a reaction with the N-methyl-2-methoxypyridinium species in the enzyme is still not reported so far. It is of great interest to know whether or not this reaction is observable in the enzyme, and more importantly, if it turns out to be not observable in the enzyme, why such a reaction proceeds quickly in aqueous solution but not in the enzyme. In the present study, we performed DFT calculations and quantum mechanical/molecular mechanical (QM/MM) calculations to reveal the fundamental mechanism for the methylation of both the methyl methane-phosphonate monoanion and the aged sarin-AChE adduct by N-methyl-2-methoxypyridinium species, respectively. The obtained results support the SN2 reaction mechanism, not the stepwise mechanism, for the methylation of the methyl methane-phosphonate monoanion by 9 reported N-methyl-2-methoxypyridinium compounds. The calculated free energy barriers are in good agreement with the experimental data. The methylation of the aged sarin-AChE adduct by one N-methyl-2-methoxypyridinium compound (labeled as compound 2) also employs the SN2 reaction mechanism with an extremely high free energy

  13. How does huperzine A enter and leave the binding gorge of acetylcholinesterase? Steered molecular dynamics simulations.

    PubMed

    Xu, Yechun; Shen, Jianhua; Luo, Xiaomin; Silman, Israel; Sussman, Joel L; Chen, Kaixian; Jiang, Hualiang

    2003-09-17

    The entering and leaving processes of Huperzine A (HupA) binding with the long active-site gorge of Torpedo californica acetylcholinesterase (TcAChE) have been investigated by using steered molecular dynamics simulations. The analysis of the force required along the pathway shows that it is easier for HupA to bind to the active site of AChE than to disassociate from it, which for the first time interprets at the atomic level the previous experimental result that unbinding process of HupA is much slower than its binding process to AChE. The direct hydrogen bonds, water bridges, and hydrophobic interactions were analyzed during two steered molecular dynamics (SMD) simulations. Break of the direct hydrogen bond needs a great pulling force. The steric hindrance of bottleneck might be the most important factor to produce the maximal rupture force for HupA to leave the binding site but it has a little effect on the binding process of HupA with AChE. Residue Asp72 forms a lot of water bridges with HupA leaving and entering the AChE binding gorge, acting as a clamp to take out HupA from or put HupA into the active site. The flip of the peptide bond between Gly117 and Gly118 has been detected during both the conventional MD and SMD simulations. The simulation results indicate that this flip phenomenon could be an intrinsic property of AChE and the Gly117-Gly118 peptide bond in both HupA bound and unbound AChE structures tends to adopt the native enzyme structure. At last, in a vacuum the rupture force is increased up to 1500 pN while in water solution the greatest rupture force is about 800 pN, which means water molecules in the binding gorge act as lubricant to facilitate HupA entering or leaving the binding gorge.

  14. 3-Oxoisoxazole-2(3H)-carboxamides and isoxazol-3-yl carbamates: Resistance-breaking acetylcholinesterase inhibitors targeting the malaria mosquito, Anopheles gambiae

    PubMed Central

    Verma, Astha; Wong, Dawn M.; Islam, Rafique; Tong, Fan; Ghavami, Maryam; Mutunga, James M.; Slebodnick, Carla; Li, Jianyong; Viayna, Elisabet; Lam, Polo C.-H.; Totrov, Maxim M.; Bloomquist, Jeffrey R.; Carlier, Paul R.

    2015-01-01

    To identify potential selective and resistance-breaking mosquitocides against the African malaria vector Anopheles gambiae, we investigated the acetylcholinesterase (AChE) inhibitory and mosquitocidal properties of isoxazol-3-yl dimethylcarbamates (15), and the corresponding 3-oxoisoxazole-2(3H)-dimethylcarboxamide isomers (14). In both series, compounds were found with excellent contact toxicity to wild-type susceptible (G3) strain and multiply resistant (Akron) strain mosquitoes that carry the G119S resistance mutation of AChE. Compounds possessing good to excellent toxicity to Akron strain mosquitoes inhibit the G119S mutant of An. gambiae AChE (AgAChE) with ki values at least 10- to 600-fold higher than that of propoxur, a compound that does not kill Akron mosquitoes at the highest concentration tested. On average, inactivation of WT AgAChE by dimethylcarboxamides 14 was 10-20 fold faster than that of the corresponding isoxazol-3-yl dimethylcarbamates 15. X-ray crystallography of dimethylcarboxamide 14d provided insight into that reactivity, a finding that may explain the inhibitory power of structurally-related inhibitors of hormone-sensitive lipase. Finally, human/An. gambiae AChE inhibition selectivities of these compounds were low, suggesting the need for additional structural modification. PMID:25684426

  15. Interaction of the collagen-like tail of asymmetric acetylcholinesterase with heparin depends on triple-helical conformation, sequence and stability.

    PubMed Central

    Deprez, P; Doss-Pepe, E; Brodsky, B; Inestrosa, N C

    2000-01-01

    The collagen-like tail of asymmetric acetylcholinesterase (AChE) contains two heparin-binding domains (HBDs) that interact with heparan sulphate proteoglycans, determining the anchoring of the enzyme at the basal lamina and its specific localization at the neuromuscular junction. Both HBDs are characterized by a cluster of basic residues containing a core with the BBXB consensus sequence (where B represents a basic residue and X a non-basic residue). To study the interaction of such HBDs with heparin we have used synthetic peptides to model the N-terminal and C-terminal sites. CD spectroscopy showed that all peptides are triple-helical at low temperatures, and undergo trimer-to-monomer transitions. Displacement assays of asymmetric AChE bound to heparin were performed using the peptides in both monomeric and triple-helical states. In the monomeric conformation, all the peptides were able to displace low levels of AChE depending on the basic charge content. In the triple-helical conformation, peptides containing the consensus sequence showed a large increase in the ability to displace bound AChE. Results suggest that the specific binding of the collagen-like-tail peptides to heparin depends both on the presence of the core sequence and on the triple-helical conformation. Moreover, BBXB-containing peptides that are less stable are more effective in displacing AChE, suggesting that the interaction region needs a significant amount of structural flexibility to better accommodate the ligand. PMID:10926855

  16. Acetylcholinesterase inhibition in cognition-relevant brain areas of mice treated with a nootropic Amazonian herbal (Marapuama).

    PubMed

    Figueiró, M; Ilha, J; Pochmann, D; Porciúncula, L O; Xavier, L L; Achaval, M; Nunes, D S; Elisabetsky, E

    2010-10-01

    The goal of acetylcholinesterase inhibitors (AChEIs) used to treat Alzheimer's patients is an improvement in cholinergic transmission. While currently available AChEIs have limited success, a huge impediment to the development of newer ones is access to the relevant brain areas. Promnesic, anti-amnesic and AChEI properties were identified in a standardized ethanol extract from Ptychopetalum olacoides (POEE), a medicinal plant favored by the elderly in Amazon communities. The purpose of this study was to provide conclusive evidence that orally given POEE induces AChE inhibition in brain areas relevant to cognition. Histochemistry experiments confirmed that the anticholinesterase compound(s) present in POEE are orally bioavailable, inducing meaningful AChE inhibition in the hippocampus CA1 (∼33%) and CA3 (∼20%), and striatum (∼17%). Ellman's colorimetric analysis revealed that G1 and G4 AChE isoforms activities were markedly inhibited (66 and 72%, respectively) in hippocampus and frontal cortex (50 and 63%, respectively), while G4 appeared to be selectively inhibited (72%) in the striatum. Western blotting showed that POEE did not induce significant changes in the AChE immunocontent suggesting that its synthesis is not extensively modified. This study provides definitive proof of meaningful anticholinesterase activity compatible with the observed promnesic and anti-amnesic effects of POEE in mice, reaffirming the potential of this extract for treating neurodegenerative conditions where a hypofunctioning cholinergic neurotransmission is prominent. Adequate assessment of the safety and efficacy of this extract and/or its isolated active compound(s) are warranted.

  17. A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime, and K048) in vivo detected by biochemical and histochemical techniques.

    PubMed

    Bajgar, Jiri; Hajek, Petr; Zdarova, Jana Karasova; Kassa, Jiri; Paseka, Antonin; Slizova, Dasa; Krs, Otakar; Kuca, Kamil; Jun, Daniel; Fusek, Josef; Capek, Lukas

    2010-12-01

    Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.

  18. Catalytic-site conformational equilibrium in nerve-agent adducts of acetylcholinesterase: possible implications for the HI-6 antidote substrate specificity.

    PubMed

    Artursson, Elisabet; Andersson, Per Ola; Akfur, Christine; Linusson, Anna; Börjegren, Susanne; Ekström, Fredrik

    2013-05-01

    Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE (mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE (hAChE) with an IC50 value of 300μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.

  19. Paper-based fluorescent sensor for rapid naked-eye detection of acetylcholinesterase activity and organophosphorus pesticides with high sensitivity and selectivity.

    PubMed

    Chang, Jiafu; Li, Haiyin; Hou, Ting; Li, Feng

    2016-12-15

    Various strategies have been proposed for the sensing of acetylcholinesterase (AChE) activity and organophosphorus pesticides (OPs). However, the practical application of most methods is restricted by their intrinsic drawbacks such as complexity, long analysis time, and high cost. Thus, it is highly desirable to develop simple, fast and sensitive approaches for AChE activity and OPs detection. Herein, we reported a simple paper-based fluorescent sensor (PFS) based on the aggregation induced emission (AIE) effect of tetraphenylethylene (TPE) and the addition reaction capability of maleimide, which has been used as a powerful tool for rapid naked-eye detection of AChE activity and OPs. The introduction of TPE provides the probe with unique fluorescence property in solid state and is of great importance for improving the sensitivity of PFS. The hydrolysis product of acetylthiocholine catalyzed by AChE induced the maleimide ring destruction and activated the fluorescence performance of TPE. Given that AChE activity can be specifically inhibited by OPs, the as-proposed PFS can also be utilized for sensitive detection of OPs. Meanwhile, the variation of fluorescence signal can be readily detected by naked eyes, and low detection limits of 2.5mUmL(-1) and 0.5ngmL(-1) for AChE activity and OPs are obtained, respectively. Moreover, it has been successfully applied for AChE activity and OPs detection in diluted human serum samples, showing its great potential to be applied in real samples. Thus, this strategy possesses considerable advantages of simplicity, rapid detection, portability, cost efficiency and visualization.

  20. Validating the importance of two acetylcholinesterases in insecticide sensitivities by RNAi in Pardosa pseudoannulata, an important predatory enemy against several insect pests.

    PubMed

    Meng, Xiangkun; Li, Chunrui; Bao, Haibo; Fang, Jichao; Liu, Zewen; Zhang, Yixi

    2015-11-01

    The pond wolf spider (Pardosa pseudoannulata) is an important predatory enemy against several insect pests and showed relative different sensitivities to organophosphate and carbamate insecticides compared to insect pests. In our previous studies, two acetylcholinesterases were identified in P. pseudoannulata and played important roles in insecticide sensitivities. In order to understand the contributions of the two acetylcholinesterases to insecticide sensitivities, we firstly employed the RNAi technology in the spider. For a suitable microinjection RNAi method, the injection site, injection volume and interference time were optimized, which then demonstrated that the injection RNAi method was applicable in this spider. With the new RNAi method, it was revealed that both Pp-AChE1 and Pp-AChE2, encoded by genes Ppace1 and Ppace2, were the targets of organophosphate insecticides, but Pp-AChE1 would be more important. In contrast, the carbamate acted selectively on Pp-AChE1. The results showed that Pp-AChE1 was the major catalytic enzyme in P. pseudoannulata and the major target of organophosphate and carbamate insecticides. In a word, an RNAi method was established in the pond wolf spider, which further validated the importance of two acetylcholinesterases in insecticide sensitivities in this spider.

  1. 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

  2. Bis(9)-(-)-nor-meptazinol as a novel dual-binding AChEI potently ameliorates scopolamine-induced cognitive deficits in mice.

    PubMed

    Liu, Ting; Xia, Zheng; Zhang, Wei-Wei; Xu, Jian-rong; Ge, Xin-Xing; Li, Juan; Cui, Yongyao; Qiu, Zhui-Bai; Xu, Jun; Xie, Qiong; Wang, Hao; Chen, Hong-Zhuan

    2013-03-01

    Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder which is characterized by the progressive deterioration of cognition and the emergence of behavioral and psychological symptoms in aging patients. Given that the clinical effectiveness of acetylcholinesterase inhibitors (AChEIs) has still been questioned due to dubious disease-modifying effects, the multi-target directed ligand (MTDL) design has become an emerging strategy for developing new drugs for AD treatment. Bis(9)-(-)-nor-meptazinol (Bis-Mep) was firstly reported by us as a novel MTDL for both potent cholinesterase and amyloid-β aggregation inhibition. In this study, we further explored its AChE inhibition kinetic features and cognitive amelioration. Bis-Mep was found to be a mixed-type inhibitor on electric eel AChE by enzyme kinetic study. Molecular docking revealed that two "water bridges" located at the two wings of Bis-Mep stabilized its interaction with both catalytic and peripheral anionic sites of AChE. Furthermore, subcutaneous administration of Bis-Mep (10, 100 or 1000 ng/kg) significantly reversed the scopolamine-induced memory deficits in a typical bell-shaped dose-response manner. The maximal cognitive amelioration of Bis-Mep was achieved at 100 ng/kg, comparable with the effect of a reference drug Huperzine A at 1 mg/kg and also the relevant AChE inhibition in brain. These findings suggested that Bis-Mep might be a promising dual-binding AChE inhibitor for potential AD therapeutics.

  3. Inhibition of rainbow trout acetylcholinesterase by aqueous and suspended particle-associated organophosphorous insecticides.

    PubMed

    Sturm, Armin; Radau, Tanja S; Hahn, Torsten; Schulz, Ralf

    2007-06-01

    Spraydrift and edge-of-field runoff are important routes of pesticide entry into streams. Pesticide contamination originating from spraydrift usually resides in the water phase, while pesticides in contaminated runoff are to a large extent associated with suspended particles (SPs). The effects of two organophosphorous insecticides (OPs), chloropyrifos (CPF) and azinphos-methyl (AZP), on acetylcholinesterase (AChE) activity in rainbow trout were compared between two exposure scenarios, simulating spraydrift- and runoff-borne contamination events in the Lourens River (LR), Western Cape, South Africa. NOECs of brain AChE inhibition, determined after 1h of exposure followed by 24h of recovery, were 0.33microgl(-1) for aqueous CPF, 200mgkg(-1) for SP-associated CPF and 20mgkg(-1) for SP-associated AZP (at 0.5gl(-1) SP). The highest aqueous AZP concentration tested (3.3microgl(-1)) was without significant effects. Previously reported peak levels of aqueous CPF in the LR ( approximately 0.2microgl(-1)) are close to its NOEC (this study), suggesting a significant toxicological risk to fish in the LR. By contrast, reported levels of SP-associated OPs in the LR are 20-200-fold lower than their NOECs (this study). In a comparative in situ study, trout were exposed for seven days at agricultural (LR2, LR3) and upstream reference (LR1) sites. No runoff occurred during the study. Brain AChE was significantly inhibited at LR3. However, OP levels at LR3 (CPF 0.01microgl(-1); AZP 0.14microgl(-1)) were minor compared to concentrations having effects in the laboratory (see above). Additionally, muscle AChE activity was significantly higher in caged trout from LR1 than in animals maintained in laboratory tanks.

  4. Copper acutely impairs behavioral function and muscle acetylcholinesterase activity in zebrafish (Danio rerio).

    PubMed

    Haverroth, Gabriela M B; Welang, Chariane; Mocelin, Riciéri N; Postay, Daniela; Bertoncello, Kanandra T; Franscescon, Francini; Rosemberg, Denis B; Dal Magro, Jacir; Dalla Corte, Cristiane L

    2015-12-01

    Copper is a heavy metal found at relatively high concentrations in surface waters around the world. Copper is a micronutrient at low concentrations and is essential to several organisms. At higher concentrations copper can become toxic, which reveal the importance of studying the toxic effects of this metal on the aquatic life. Thus, the objective of this study was to evaluate the toxic effects of copper on the behavior and biochemical parameters of zebrafish (Danio rerio). Zebrafish were exposed for 24h at a concentration of 0.006 mg/L Cu. After the exposure period, behavioral profile of animals was recorded through 6 min using two different apparatuses tests: the Novel Tank and the Light-Dark test. After behavioral testing, animals were euthanized with a solution of 250 mg/L of tricaine (MS-222). Brain, muscle, liver and gills were extracted for analysis of parameters related to oxidative stress and accumulation of copper in these tissues. Acetylcholinesterase (AChE) activity was determined in brain and muscle. Results showed acute exposure to copper induces significant changes in behavioral profile of zebrafish by changing locomotion and natural tendency to avoid brightly lit area. On the other hand, there were no significant effects on parameters related to oxidative stress. AChE activity decreased significantly in zebrafish muscle, but there were no significant changes in cerebral AChE activity. Copper levels in tissues did not increase significantly compared to the controls. Taken together, these results indicate that a low concentration of copper can acutely affect behavioral profile of adult zebrafish which could be partially related to an inhibition on muscle AChE activity. These results reinforce the need of additional tests to establishment of safe copper concentrations to aquatic organisms and the importance of behavioral parameters in ecotoxicological studies.

  5. Isolation and characterisation of acetylcholinesterase inhibitors from Aquilaria subintegra for the treatment of Alzheimer's disease (AD).

    PubMed

    Bahrani, Hirbod; Mohamad, Jamaludin; Paydar, Mohammad Javad; Rothan, Hussin A

    2014-02-01

    Aquilaria subintegra, locally known as "Gaharu", belongs to the Thymelaeceae family. This plant's leaves have been claimed to be effective for the treatment of Alzheimer's disease (AD) by Malay traditional practitioner in Malaysia. In this research, the chloroform extracts of the leaves and stem of A. subintegra were tested for acetylcholinesterase (AChE) inhibitory activity. The Thin Layer Chromatography (TLC) results indicated the presence of phenols, flavonoids, terpenoids, and alkaloids compounds in the extracts. Analysis of the stem chloroform extracts with LCMS/MS displayed that it contains kaempferol 3,4,7-trimethyl ether. The AChE inhibitory activity of leaves and stem chloroform extracts and kaempferol were 80%, 93% and 85.8%, respectively. The Brine Shrimp Lethality Assay (BSLA) exhibited low to moderate toxicity of the chloroform extract from leaves (LC50=531.18 ± 49.53 μg/ml), the stem chloroform extract (LC50=407.34 ± 68.05 μg/ml) and kaempferol (LC50=762.41 ± 45.09 μg/ml). The extracts and kaempferol were not cytotoxic to human umbilical vein endothelial cells (HUVEC), human normal gastric epithelial cell line (GES-1) and human normal hepatic cell line (WRL-68). The effect of leaf and stem chloroform extracts and kaempferol were determined in the Radial Arm Maze (RAM) after administration by oral gavage to ICR male and female mice with valium-impaired memory. Administration of kaempferol to the mice significantly reduced the number of repeated entries into the arms of maze in males and females. In conclusion, the inhibition of AChE by leaf and stem chloroform extracts of A. subintegra could be due to the presence of kaempferol. This extract is safe for use as a natural AChE inhibitor as an alternative to berberine for the treatment of AD.

  6. Acetylcholinesterase Inhibition by Biofumigant (Coumaran) from Leaves of Lantana camara in Stored Grain and Household Insect Pests

    PubMed Central

    Raghavendra, Anjanappa; Bakthavatsalam, Nandagopal

    2014-01-01

    Recent studies proved that the biofumigants could be an alternative to chemical fumigants against stored grain insect pests. For this reason, it is necessary to understand the mode of action of biofumigants. In the present study the prospectus of utilising Lantana camara as a potent fumigant insecticide is being discussed. Inhibition of acetylcholinesterase (AChE) by Coumaran, an active ingredient extracted from the plant L. camara, was studied. The biofumigant was used as an enzyme inhibitor and acetylthiocholine iodide as a substrate along with Ellman's reagent to carry out the reactions. The in vivo inhibition was observed in both dose dependent and time dependent in case of housefly, and the nervous tissue (ganglion) and the whole insect homogenate of stored grain insect exposed to Coumaran. The possible mode of action of Coumaran as an acetylcholinesterase inhibitor is discussed. PMID:25025036

  7. Acetylcholinesterase inhibition by biofumigant (Coumaran) from leaves of Lantana camara in stored grain and household insect pests.

    PubMed

    Rajashekar, Yallappa; Raghavendra, Anjanappa; Bakthavatsalam, Nandagopal

    2014-01-01

    Recent studies proved that the biofumigants could be an alternative to chemical fumigants against stored grain insect pests. For this reason, it is necessary to understand the mode of action of biofumigants. In the present study the prospectus of utilising Lantana camara as a potent fumigant insecticide is being discussed. Inhibition of acetylcholinesterase (AChE) by Coumaran, an active ingredient extracted from the plant L. camara, was studied. The biofumigant was used as an enzyme inhibitor and acetylthiocholine iodide as a substrate along with Ellman's reagent to carry out the reactions. The in vivo inhibition was observed in both dose dependent and time dependent in case of housefly, and the nervous tissue (ganglion) and the whole insect homogenate of stored grain insect exposed to Coumaran. The possible mode of action of Coumaran as an acetylcholinesterase inhibitor is discussed.

  8. Antioxidant and anti-acetylcholinesterase activities of extracts and secondary metabolites from Acacia cyanophylla

    PubMed Central

    Ghribia, Lotfi; Ghouilaa, Hatem; Omrib, Amel; Besbesb, Malek; Janneta, Hichem Ben

    2014-01-01

    Objective To investigate the antioxidant potential and anti-acetycholinesterase activity of compounds and extracts from Acacia cyanophylla (A. cyanophylla). Methods Three polyphenolic compounds were isolated from ethyl acetate extract of A. cyanophylla flowers. They have been identified as isosalipurposide 1, quercetin 2 and naringenin 3. Their structures were elucidated by extensive spectroscopic methods including 1D and 2D NMR experiments as well as ES-MS. The prepared extracts and the isolated compounds 1-3 were tested for their antioxidant activity using 1′-1′-diphenylpicrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging assays and reducing power. They have been also investigated for inhibitory effect against acetylcholinesterase using the microplate assay. Results In the DPPH test, the EtOAc extract of flowers exhibited the highest antioxidant effect (67.26 µg/mL). Isosalipurposide 1 showed a significant antiradical power against DPPH (81.9 µg/mL). All extracts showed a dose-dependent acetylcholinesterase inhibition. In terms of the IC50 value, the butanolic extract (16.03 µg/mL) was the most potent sample. Isosalipurposide 1 was found to be active against AChE with an IC50 value of 52.04 µg/mL. Conclusions The results demonstrated the important antioxidant and anti-acetylcholinesterase activity of pure compounds and extracts from A. cyanophylla. PMID:25183120

  9. Can hydroxylamine be a more potent nucleophile for the reactivation of tabun-inhibited AChE than prototype oxime drugs? An answer derived from quantum chemical and steered molecular dynamics studies.

    PubMed

    Lo, Rabindranath; Ganguly, Bishwajit

    2014-07-29

    Organophosphorus nerve agents are highly toxic compounds which strongly inhibit acetylcholinesterase (AChE) in the blood and in the central nervous system (CNS). Tabun is one of the highly toxic organophosphorus (OP) compounds and is resistant to many oxime drugs formulated for the reactivation of AChE. The reactivation mechanism of tabun-conjugated AChE with various drugs has been examined with density functional theory and ab initio quantum chemical calculations. The presence of a lone-pair located on the amidic group resists the nucleophilic attack at the phosphorus center of the tabun-conjugated AChE. We have shown that the newly designed drug candidate N-(pyridin-2-yl)hydroxylamine, at the MP2/6-31+G*//M05-2X/6-31G* level in the aqueous phase with the polarizable continuum solvation model (PCM), is more effective in reactivating the tabun-conjugated AChE than typical oxime drugs. The rate determining activation barrier with N-(pyridin-2-yl)hydroxylamine was found to be ∼1.7 kcal mol(-1), which is 7.2 kcal mol(-1) lower than the charged oxime trimedoxime (one of the most efficient reactivators in tabun poisonings). The greater nucleophilicity index (ω(-)) and higher CHelpG charge of pyridinylhydroxylamine compared to TMB4 support this observation. Furthermore, we have also examined the reactivation process of tabun-inhibited AChE with some other bis-quaternary oxime drug candidates such as methoxime (MMB4) and obidoxime. The docking analysis suggests that charged bis-quaternary pyridinium oximes have greater binding affinity inside the active-site gorge of AChE compared to the neutral pyridinylhydroxylamine. The peripheral ligand attached to the neutral pyridinylhydroxylamine enhanced the binding with the aromatic residues in the active-site gorge of AChE through effective π-π interactions. Steered molecular dynamics (SMD) simulations have also been performed with the charged oxime (TMB4) and the neutral hydroxylamine. From protein-drug interaction

  10. Acetylcholinesterase activity in the erythrocytes of newborn infants with hyperbilirubinemia and asphyxia.

    PubMed

    Mydlil, V; Tomasová, H; Cápová, E; Cerná, M

    1976-01-01

    The authors measured erythrocyte acetylcholinesterase (ACHE) activity in 51 physiologic newborn infants, 35 pregnant women and 101 infants with hyperbilirubinemia caused by hemolytic diseases in the ABO and Rh systems, or of other origin (6 children). In total, 374 examinations of erythrocyte ACHE were performed. The normal values of physiologic infants during the first hours of life were 0.162 +/- 0.03, at the age of 3 days 0.104 +/- 0.04. The values in pregnant women were 0.206 +/- 0.07. The ACHE activity was considerably decreased in newborn infants affected by erythroblastosis-ABO, and its mean value, in the case of exsanguinotransfusion indicated by the Polácek scheme, amounted to 0.060 +/- 0.051. It raised after exsanguinotransfusion and was equal to the value of red cells transfused. In newborn infants affected by erythroblastosis-Rh, the value decreased in the cases complicated with asphyxia. Low values of ACHE are sometimes found in certain cases of IRDS or of septicemia. The determination of erythrocyte ACHE activity enables assessing the state of red blood cells.

  11. Memory deficits correlating with acetylcholinesterase splice shift and amyloid burden in doubly transgenic mice.

    PubMed

    Rees, Tina M; Berson, Amit; Sklan, Ella H; Younkin, Linda; Younkin, Steven; Brimijoin, Stephen; Soreq, Hermona

    2005-07-01

    Current mouse models of Alzheimer's disease show brain pathology that correlates to a degree with memory impairment, but underlying molecular mechanisms remained unknown. Here we report studies with three lines of transgenic mice: animals that doubly express mutated human amyloid precursor protein (APPswe) and human acetylcholinesterase (hAChE); and animals transgenic for only the APPswe or the hAChE. Among these genotypes, variations were observed in expression of mRNA for presenilin-1, which was highest in singly transgenic hAChE mice, and the stress-inducible form of AChE, which was elevated when both transgenes were present. At the age of nine months, both double and single transgenic mice displayed working memory impairment in a radial arm water maze. However, as compared with mice expressing amyloid alone, the double transgenic animals exhibited more numerous plaques and greater amyloid burden in brain (both by histochemistry and by ELISA of amyloid protein). Moreover, the amyloid burden in double transgenics was tightly correlated with memory impairment as measured by total maze errors (r2= 0.78, p = .002). This correlation was markedly stronger than observed in mice with amyloid alone. These new findings support the notion of cholinergic-amyloid interrelationships and highlight the double transgenic mice as a promising alternative for testing Alzheimer's therapies.

  12. A Soluble Acetylcholinesterase Provides Chemical Defense against Xenobiotics in the Pinewood Nematode

    PubMed Central

    Kang, Jae Soon; Lee, Dae-Weon; Koh, Young Ho; Lee, Si Hyeock

    2011-01-01

    The pinewood nematode genome encodes at least three distinct acetylcholinesterases (AChEs). To understand physiological roles of the three pinewood nematode AChEs (BxACE-1, BxACE-2, and BxACE-3), BxACE-3 in particular, their tissue distribution and inhibition profiles were investigated. Immunohistochemistry revealed that BxACE-1 and BxACE-2 were distributed in neuronal tissues. In contrast, BxACE-3 was detected from some specific tissues and extracted without the aid of detergent, suggesting its soluble nature unlike BxACE-1 and BxACE-2. When present together, BxAChE3 significantly reduced the inhibition of BxACE-1 and BxACE-2 by cholinesterase inhibitors. Knockdown of BxACE-3 by RNA interference significantly increased the toxicity of three nematicidal compounds, supporting the protective role of BxACE-3 against chemicals. In summary, BxACE-3 appears to have a non-n