The activity of detoxifying enzymes in the infective juveniles of Heterorhabditis bacteriophora strains: Purification and characterization of two acetylcholinesterases.

PubMed

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

2016-02-01

The infectivity and detoxifying enzyme activities including glutathione-S-transferase (GST), acetylcholinesterase (AChE) and carboxylesterase (CaE) are investigated in the infective juveniles (IJs) of six different strains of Heterorhabditis bacteriophora as a biocontrol agent against insect pests. The specific activities ranged from 10.8-29.8 and 50-220units/mg protein for GST and AChE, respectively; and from 24.7-129 and 22.6-77.3units/mg protein for CaE as estimated by P-nitrophenyl and α-naphthyl acetates, respectively. H. bacteriophora EM2 strain has the highest infectivity and the highest enzymatic activities as well. AChE is the predominant detoxifying enzyme that might imply its major role in the detoxification of insecticide(s). The isoenzyme pattern demonstrated two major slow-moving isoforms in all EPN strains examined. Purification of two AChE isoforms, AChEAII and AChEBI, from H. bacteriophora EM2 strain is performed by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and chromatography on DEAE-Sepharose. AChEAII and AChEBII have specific activities of 1207 and 1560unit/mg protein, native molecular weights of 180 and 68kDa, and are found in dimeric and monomeric forms, respectively. Both isoforms showed optimum activity at pH8.5 and 35°C. AChEBI exhibited higher thermal stability and higher activation energy than AChEAII. The enzymatic activities of purified AChEs are completely inhibited by Hg(+2) and Ni(+2) and greatly enhanced by Mn(+2). The substrate specificity, the relative efficiency of substrates hydrolysis, substrate inhibition and inhibition by BW284C51, but not by iso-OMPA, clearly indicated that they are true AChEs; their properties are compared with those recorded for insects as target hosts for H. bacteriophora EM2. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Phytochemical and ethnomedicinal study of Huperzia species used in the traditional medicine of Saraguros in Southern Ecuador; AChE and MAO inhibitory activity.

PubMed

Armijos, Chabaco; Gilardoni, Gianluca; Amay, Luis; Lozano, Antonio; Bracco, Francesco; Ramirez, Jorge; Bec, Nicole; Larroque, Christian; Finzi, Paola Vita; Vidari, Giovanni

2016-12-04

This study concerns seven Huperzia species (Lycopodiaceae), namely H. brevifolia, H. columnaris, H. compacta, H. crassa, H. espinosana, H. tetragona, H. weberbaueri, which are considered sacred plants by the Saraguro community, living in the Southern Andes of Ecuador; these plants are widely used in traditional medicine and ritual ceremonies. The plants were selected on the basis of written interviews with 10 visionary healers (yachak) (2 women, 8 men), indicated as the most credible by the Saraguro Healers Council. The Informant Consensus Factor (F ic ) was determined. The first phytochemical study of the plants was performed by standard procedures, while the AChE and MAO-A inhibition by fractions enriched in high MW alkaloids, was measured in vitro. i) to investigate the uses of some Huperzia plants in healing and magical-religious practices of Saraguros; ii) to identify the main components of plant hydromethanolic extracts; iiì) to test the effects of alkaloidal fractions on the activity of two enzymes linked to mental health. All the interviewed Saraguro yachak showed a high consensus about the uses of the seven Huperzia plants as purgatives and against supernatural diseases, such as the "espanto" (startle). In admixtures with other plants, some species also induce a state of trance or hallucinations in participants in magical-religious rituals. GC-MS of the volatile alkaloid fractions allowed the identification of some lycodine-type and lycopodine-type alkaloids (1-5) in H. compacta, H. columnaris, and H. tetragona. The flavones selgin) (6) and tricin (7) were isolated from H. brevifolia and H. espinosana. Tricin (7) was also detected in the other five species. The rare serratene triterpenes serratenediol (8) serratenediol-3-O-acetate (9), 21-episerratenediol (10), and 21-episerratenediol-3-O-acetate (11) were isolated from H. crassa. In addition, the presence of an unprecedented group of high molecular weight alkaloids has been determined. Alkaloid fractions

Chlorpyrifos and malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity.

PubMed

Richendrfer, Holly; Creton, Robbert

2015-07-01

Organophosphates, a type of neurotoxicant pesticide, are used globally for the treatment of pests on croplands and are therefore found in a large number of conventional foods. These pesticides are harmful and potentially deadly if ingested or inhaled in large quantities by causing a significant reduction in acetylcholinesterase (AChE) activity in the central and peripheral nervous system. However, much less is known about the effects of exposure to small quantities of the pesticides on neural systems and behavior during development. In the current study we used zebrafish larvae in order to determine the effects of two of the most widely used organophosphates, chlorpyrifos and malathion, on zebrafish behavior and AChE activity. Embryos and larvae were exposed to the organophosphates during different time points in development and then tested at 5 days post-fertilization for behavioral, neurodevelopmental and AChE abnormalities. The results of the study indicate that chlorpyrifos and malathion cause opposing behaviors in the larvae such as swim speed (hypoactivity vs. hyperactivity) and rest. Additionally, the pesticides affect only certain behaviors, such as thigmotaxis, during specific time points in development that are unrelated to changes in AChE activity. Larvae treated with malathion but not chlorpyrifos also had significantly smaller forebrain and hindbrain regions compared to controls by 5 days post-fertilization. We conclude that exposure to very low concentrations of organophosphate pesticides during development cause abnormalities in behavior and brain size. Copyright © 2015 Elsevier Inc. All rights reserved.

Chlorpyrifos and Malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity

PubMed Central

Richendrfer, Holly; Creton, Robbert

2015-01-01

Organophosphates, a type of neurotoxicant pesticide, are used globally for the treatment of pests on croplands and are therefore found in a large number of conventional foods. These pesticides are harmful and potentially deadly if ingested or inhaled in large quantities by causing a significant reduction in acetylcholinesterase (AChE) activity in the central and peripheral nervous system. However, much less is known about the effects of exposure to small quantities of the pesticides on neural systems and behavior during development. In the current study we used zebrafish larvae in order to determine the effects of two of the most widely used organophosphates, chlorpyrifos and malathion, on zebrafish behavior and AChE activity. Embryos and larvae were exposed to the organophosphates during different time points in development and then tested at 5 days post-fertilization for behavioral, neurodevelopmental and AChE abnormalities. The results of the study indicate that chlorpyrifos and malathion cause opposing behaviors in the larvae such as swim speed (hypoactivity vs. hyperactivity) and rest. Additionally, the pesticides affect only certain behaviors, such as thigmotaxis, during specific time points in development that are unrelated to changes in AChE activity. Larvae treated with malathion but not chlorpyrifos also had significantly smaller forebrain and hindbrain regions compared to controls by 5 days post-fertilization. We conclude that exposure to very low concentrations of organophosphate pesticides during development cause abnormalities in behavior and brain size. PMID:25983063

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.

A facile stereoselective synthesis of dispiro-indeno pyrrolidine/pyrrolothiazole-thiochroman hybrids and evaluation of their antimycobacterial, anticancer and AchE inhibitory activities.

PubMed

Bharkavi, Chelliah; Vivek Kumar, Sundaravel; Ashraf Ali, Mohamed; Osman, Hasnah; Muthusubramanian, Shanmugam; Perumal, Subbu

2016-11-15

A facile stereoselective synthesis of novel dispiro indeno pyrrolidine/pyrrolothiazole-thiochroman hybrids has been achieved by 1,3-dipolar cycloaddition of azomethine ylides, generated in situ from ninhydrin and sarcosine/thiaproline, on a series of 3-benzylidenethiochroman-4-ones. The synthesised compounds were screened for their antimycobacterial, anticancer and AchE inhibition activities. Compound 4l (IC 50 1.07μM) has been found to exhibit the most potent antimycobacterial activity compared to cycloserine (12 times), pyrimethamine (37 times) and ethambutol (IC 50 <1.56μM) and 6l (IC 50 =2.87μM) is more active than both cycloserine (4 times) and pyrimethamine (12 times). Three compounds, 4a, 6b and 6i, display good anticancer activity against CCRF-CEM cell lines. Compounds 6g and 4g display maximum AchE inhibitory activity with IC 50 values of 1.10 and 1.16μmol/L respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of enzymes inhibition activities of medicinal plant from Burkina Faso.

PubMed

Bangou, Mindiédiba Jean; Kiendrebeogo, Martin; Meda, Nâg-Tiero Roland; Coulibaly, Ahmed Yacouba; Compaoré, Moussa; Zeba, Boukaré; Millogo-Rasolodimby, Jeanne; Nacoulma, Odile Germaine

2011-01-15

The aim of the present study was to evaluate some enzymes inhibitory effects of 11 plant species belonging to 9 families from Burkina Faso. Methanolic extracts were used for their Glutathione-s-transferase (GST), Acetylcholinesterase (AChE), Carboxylesterase (CES) and Xanthine Oxidase (XO) inhibitory activities at final concentration of 100 microg mL(-1). The total phenolics, flavonoids and tannins were also determined spectrophotometrically using Folin-Ciocalteu, AlCl3 and ammonium citrate iron reagents, respectively. Among the 11 species tested, the best inhibitory percentages were found with Euphorbia hirta, Sclerocarya birrea and Scoparia dulcis (inhibition > 40%) followed by Annona senegalensis, Annona squamosa, Polygala arenaria and Ceratotheca sesamoides (inhibition > 25%). The best total phenolic and tannin contents were found with S. birrea with 56.10 mg GAE/100 mg extract and 47.75 mg TAE/100 mg extract, respectively. E hirta presented the higher total flavonoids (9.96 mg QE/100 mg extract). It's was found that Sclerocarya birrea has inhibited all enzymes at more than 30% and this activity is correlated to total tannins contents. Contrary to S. birrea, the enzymatic activities of E. hirta and S. dulcis are correlated to total flavonoids contents. Present findings suggest that the methanolic extracts of those plant species are potential inhibitors of GST, AChE, CES and XO and confirm their traditional uses in the treatment of mental disorders, gout, painful inflammations and cardiovascular diseases.

Inhibitory effects of oxytocin and oxytocin receptor antagonist atosiban on the activities of carbonic anhydrase and acetylcholinesterase enzymes in the liver and kidney tissues of rats.

PubMed

Kocyigit, Umit M; Taşkıran, Ahmet Şevki; Taslimi, Parham; Yokuş, Ahmet; Temel, Yusuf; Gulçin, İlhami

2017-11-01

The aim of this study was to investigate the effects of oxytocin (OT), atosiban, which is an OT receptor antagonist, and OT-atosiban chemicals injected to rats on the activities of carbonic anhydrase (CA) and acetylcholinesterase (AChE) enzymes in liver and kidney tissues of rats. For this purpose, four different groups, each consisting of six rats (n = 6), were formed (control group, OT administered group, atosiban administered group, and both OT and atosiban administered group). The rats were necropsied 60 min after intraperitoneal injection of chemicals into the rats. Liver tissues of rats were extracted. CA and AChE enzyme activities were measured for each tissue by using hydratase, esterase, and acetylcholiniodide methods. Activity values for each enzyme obtained were statistically calculated. © 2017 Wiley Periodicals, Inc.

In-silico identification of the binding mode of synthesized adamantyl derivatives inside cholinesterase enzymes

PubMed Central

Al-Aboudi, Amal; Al-Qawasmeh, Raed A; Shahwan, Alaa; Mahmood, Uzma; Khalid, Asaad; Ul-Haq, Zaheer

2015-01-01

Aim: To investigate the binding mode of synthesized adamantly derivatives inside of cholinesterase enzymes using molecular docking simulations. Methods: A series of hybrid compounds containing adamantane and hydrazide moieties was designed and synthesized. Their inhibitory activities against acetylcholinesterase (AChE) and (butyrylcholinesterase) BChE were assessed in vitro. The binding mode of the compounds inside cholinesterase enzymes was investigated using Surflex-Dock package of Sybyl7.3 software. Results: A total of 26 adamantyl derivatives were synthesized. Among them, adamantane-1-carboxylic acid hydrazide had an almost equal inhibitory activity towards both enzymes, whereas 10 other compounds exhibited moderate inhibitory activity against BChE. The molecular docking studies demonstrated that hydrophobic interactions between the compounds and their surrounding residues in the active site played predominant roles, while hydrophilic interactions were also found. When the compounds were docked inside each enzyme, they exhibited stronger interactions with BChE over AChE, possibly due to the larger active site of BChE. The binding affinities of the compounds for BChE and AChE estimated were in agreement with the experimental data. Conclusion: The new adamantly derivatives selectively inhibit BChE with respect to AChE, thus making them good candidates for testing the hypothesis that BChE inhibitors would be more efficient and better tolerated than AChE inhibitors in the treatment of Alzheimer's disease. PMID:25937631

Carnitine modulates crucial myocardial adenosine triphosphatases and acetylcholinesterase enzyme activities in choline-deprived rats.

PubMed

Strilakou, Athina A; Tsakiris, Stylianos T; Kalafatakis, Konstantinos G; Stylianaki, Aikaterini T; Karkalousos, Petros L; Koulouris, Andreas V; Mourouzis, Iordanis S; Liapi, Charis A

2014-01-01

Choline is an essential nutrient, and choline deficiency has been associated with cardiovascular morbidity. Choline is also the precursor of acetylcholine (cholinergic component of the heart's autonomic nervous system), whose levels are regulated by acetylcholinesterase (AChE). Cardiac contraction-relaxation cycles depend on ion gradients established by pumps like the adenosine triphosphatases (ATPases) Na(+)/K(+)-ATPase and Mg(2+)-ATPase. This study aimed to investigate the impact of dietary choline deprivation on the activity of rat myocardial AChE (cholinergic marker), Na(+)/K(+)-ATPase, and Mg(2+)-ATPase, and the possible effects of carnitine supplementation (carnitine, structurally relevant to choline, is used as an adjunct in treating cardiac diseases). Adult male albino Wistar rats were distributed among 4 groups, and were fed a standard or choline-deficient diet for one month with or without carnitine in their drinking water (0.15% w/v). The enzyme activities were determined spectrophotometrically in the myocardium homogenate. Choline deficiency seems to affect the activity of the aforementioned parameters, but only the combination of choline deprivation and carnitine supplementation increased myocardial Na(+)/K(+)-ATPase activity along with a concomitant decrease in the activities of Mg(2+)-ATPase and AChE. The results suggest that carnitine, in the setting of choline deficiency, modulates cholinergic myocardial neurotransmission and the ATPase activity in favour of cardiac work efficiency.

Association of health symptoms with low-level exposure to organophosphates, DNA damage, AChE activity, and occupational knowledge and practice among rice, corn, and double-crop farmers

PubMed Central

Hongsibsong, Surat; Sittitoon, Nalin; Sapbamrer, Ratana

2017-01-01

Objectives: This study aims to determine (1) total dialkylphosphate (ΣDAP) levels, occupational knowledge and practice, DNA damage, AChE activity, and health symptoms in rice, corn, and double-crop farmers; (2) the association of health symptoms with ΣDAP levels, occupational knowledge and practice, DNA damage, and AChE activity in farmers; and (3) the prevalence of health symptoms between farmers and non-farmers. Methods: A cross-sectional study was conducted by interviewing as well as analyzing urine and blood samples during July to August 2014. Results: There were no differences in ΣDAP levels, AChE activity, and occupational knowledge and practice scores among all farmer groups. In terms of health symptoms related to ΣDAP, AChE activity, DNA damage, and occupational knowledge and practice, pesticide-related symptoms were determined, including breathlessness, chest pain, dry throat, numbness, muscle weakness, cramp, headache, dizziness, eye irritation, white/red rash, and white/red pimple, which were classified as respiratory, muscle, nervous, and epithelial symptoms. A remarkable finding was that farmers had a significantly higher prevalence of muscle weakness (odds ratio (OR)=3.79) and numbness (OR=3.45) as compared with non-farmers. Conclusion: Our findings, therefore, suggest that a long-term low-level exposure to organophosphates (OPs) may be associated with an increasing prevalence of muscle symptoms. However, a further cohort study incorporating sensitive health outcomes and measurement of multiple pesticides monitoring on a larger scale is warranted. PMID:28077823

Association of health symptoms with low-level exposure to organophosphates, DNA damage, AChE activity, and occupational knowledge and practice among rice, corn, and double-crop farmers.

PubMed

Hongsibsong, Surat; Sittitoon, Nalin; Sapbamrer, Ratana

2017-03-28

This study aims to determine (1) total dialkylphosphate (ΣDAP) levels, occupational knowledge and practice, DNA damage, AChE activity, and health symptoms in rice, corn, and double-crop farmers; (2) the association of health symptoms with ΣDAP levels, occupational knowledge and practice, DNA damage, and AChE activity in farmers; and (3) the prevalence of health symptoms between farmers and non-farmers. A cross-sectional study was conducted by interviewing as well as analyzing urine and blood samples during July to August 2014. There were no differences in ΣDAP levels, AChE activity, and occupational knowledge and practice scores among all farmer groups. In terms of health symptoms related to ΣDAP, AChE activity, DNA damage, and occupational knowledge and practice, pesticide-related symptoms were determined, including breathlessness, chest pain, dry throat, numbness, muscle weakness, cramp, headache, dizziness, eye irritation, white/red rash, and white/red pimple, which were classified as respiratory, muscle, nervous, and epithelial symptoms. A remarkable finding was that farmers had a significantly higher prevalence of muscle weakness (odds ratio (OR)=3.79) and numbness (OR=3.45) as compared with non-farmers. Our findings, therefore, suggest that a long-term low-level exposure to organophosphates (OPs) may be associated with an increasing prevalence of muscle symptoms. However, a further cohort study incorporating sensitive health outcomes and measurement of multiple pesticides monitoring on a larger scale is warranted.

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.

The structure-AChE inhibitory activity relationships study in a series of pyridazine analogues.

PubMed

Saracoglu, M; Kandemirli, F

2009-07-01

The structure-activity relationships (SAR) are investigated by means of the Electronic-Topological Method (ETM) followed by the Neural Networks application (ETM-NN) for a class of anti-cholinesterase inhibitors (AChE, 53 molecules) being pyridazine derivatives. AChE activities of the series were measured in IC(50) units, and relative to the activity levels, the series was partitioned into classes of active and inactive compounds. Based on pharmacophores and antipharmacophores calculated by the ETM-software as sub-matrices containing important spatial and electronic characteristics, a system for the activity prognostication is developed. Input data for the ETM were taken as the results of conformational and quantum-mechanics calculations. To predict the activity, we used one of the most well known neural networks, namely, the feed-forward neural networks (FFNNs) trained with the back propagation algorithm. The supervised learning was performed using a variant of FFNN known as the Associative Neural Networks (ASNN). The result of the testing revealed that the high ETM's ability of predicting both activity and inactivity of potential AChE inhibitors. Analysis of HOMOs for the compounds containing Ph1 and APh1 has shown that atoms with the highest values of the atomic orbital coefficients are mainly those atoms that enter into the pharmacophores. Thus, the set of pharmacophores and antipharmacophores found as the result of this study forms a basis for a system of the anti-cholinesterase activity prediction.

Activity changes of antioxidant and detoxifying enzymes in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae infected by the entomopathogenic nematode Heterorhabditis beicherriana (Rhabditida: Heterorhabditidae).

PubMed

Li, Xingyue; Liu, Qizhi; Lewis, Edwin E; Tarasco, Eustachio

2016-12-01

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis are lethal parasites of many insect species. To investigate defensive mechanisms towards EPNs in relation to antioxidative and detoxifying enzymes, we chose Tenebrio molitor (Coleoptera: Tenebrionidae) as experimental insect. We studied the activity changes of superoxide dismutases (SODs), peroxidases (PODs), and catalases (CATs), as well as tyrosinase (TYR), acetylcholinesterase (AChE), carboxylesterase (CarE), and glutathione S-transferase (GSTs) for 40 h in T. molitor larvae infected with Heterorhabditis beicherriana infective juveniles (IJs) at 5 rates (0, 20, 40, 80, and 160 IJs/larva). We found that when T. molitor larvae infected with H. beicherriana at higher rates (80 and 160 IJs/larva), SOD activity quickly increased to more than 70 % higher than that control levels. The activities of POD and CAT increased after 24 h. TYR activity increased slowly at lower rates of infection for 16 h, followed by a slight decrease, and then increasing from 32 to 40 h. The other detoxifying enzymes (GST, CarE, and AChE) were enhanced at lower infection rates, but were inhibited at higher rates. Our results suggested that host antioxidative response and detoxification reactions played a central role in the defensive reaction to EPNs, and that this stress which was reflected by the higher level enzymes activity contributed to the death of hosts. Further study should explore the exact function of these enzymes using different species of EPNs and investigate the links between enzyme activity and host susceptibility to EPNs.

Studies on the interaction of BDE-47 and BDE-209 with acetylcholinesterase (AChE) based on the neurotoxicity through fluorescence, UV-vis spectra, and molecular docking.

PubMed

Wang, Shutao; Wu, Chuan; Liu, Zhisheng; You, Hong

2018-05-01

The neurotoxicity of polybrominated diphenyl ethers (PBDEs) has been of concern. Acetylcholinesterase (AChE) is a critical enzyme in the central and peripheral nervous system related to neurotoxicity. The interaction between BDE-47, BDE-209, and AChE was investigated through fluorescence and UV-vis spectra combined with molecular docking. Both BDE-47 and BDE-209 bound with AChE and changed the microenvironment of some amino acid residues, resulting in a change of AChE conformation. Hydrophobic interaction is the main binding force between BDE-47, BDE-209, and AChE, and electrostatic interaction exists according to the thermodynamic parameters of the interaction between them. A hydrophobic interaction of BDE-47-AChE and BDE-209-AChE has been confirmed through molecular docking to dominate the binding force. The binding constants of BDE-47-AChE and BDE-209-AChE were 4.2 × 10 4 and 4.1 × 10 4  L/mol, respectively, and the lowest binding energies of BDE-47-AChE and BDE-209-AChE were -7.8 and -5.9 kJ/mol, respectively. BDE-47 is more likely to bind with AChE than BED-209. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis and discovery of highly functionalized mono- and bis-spiro-pyrrolidines as potent cholinesterase enzyme inhibitors.

PubMed

Kia, Yalda; Osman, Hasnah; Suresh Kumar, Raju; Basiri, Alireza; Murugaiyah, Vikneswaran

2014-04-01

Novel mono and bis spiropyrrolidine derivatives were synthesized via an efficient ionic liquid mediated, 1,3-dipolar cycloaddition methodology and evaluated in vitro for their AChE and BChE inhibitory activities in search for potent cholinesterase enzyme inhibitors. Most of the synthesized compounds displayed remarkable AChE inhibitory activities with IC50 values ranging from 1.68 to 21.85 μM, wherein compounds 8d and 8j were found to be most active inhibitors against AChE and BChE with IC50 values of 1.68 and 2.75 μM, respectively. Molecular modeling simulation on Torpedo californica AChE and human BChE receptors, showed good correlation between IC50 values and binding interaction template of the most active inhibitors docked into the active site of their relevant enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative investigation of two methods for Acetylcholinesterase enzyme immobilization on modified porous silicon

NASA Astrophysics Data System (ADS)

Khaldi, Khadidja; Sam, Sabrina; Lounas, Amel; Yaddaden, Chafiaa; Gabouze, Noure-Eddine

2017-11-01

In this work, Acetylcholinesterase enzyme (AChE) was immobilized on porous silicon (PSi) surface using two strategies. In the first method, acid chains were covalently grafted on the hydrogenated PSi by hydrosilylation reaction. The obtained acid-terminated surface was activated by a reaction with N-hydroxysuccinimide (NHS) in the presence of a peptide-coupling agent N-ethyl-N‧-(3-dimethylaminopropyl)-carbodiimide (EDC), and then reacted with the amino linker of the lysine residues AChE to anchor the enzyme by a covalent amide bond. In the second procedure, the PSi surface was first hydroxylated in piranha solution, followed by a silanization reaction with 3-aminopropyltriethoxysilane (APTES) to form amine-terminated surface. Finally, AChE was attached to the terminal amine groups by an aminolysis reaction with carboxylic acid groups of AChE in the presence of NHS/EDC mixture. Fourier transform infrared spectroscopy (FTIR) confirmed the efficiency of the surface modifications. The enzymatic activity of immobilized AChE was determined by means of a colorimetric test and was discussed according to the enzyme orientation on the surface which was revealed by contact angle measurements.

Assessing the reactivation efficacy of hydroxylamine anion towards VX-inhibited AChE: a computational study.

PubMed

Khan, Md Abdul Shafeeuulla; Ganguly, Bishwajit

2012-05-01

Oximate anions are used as potential reactivating agents for OP-inhibited AChE because of they possess enhanced nucleophilic reactivity due to the α-effect. We have demonstrated the process of reactivating the VX-AChE adduct with formoximate and hydroxylamine anions by applying the DFT approach at the B3LYP/6-311 G(d,p) level of theory. The calculated results suggest that the hydroxylamine anion is more efficient than the formoximate anion at reactivating VX-inhibited AChE. The reaction of formoximate anion and the VX-AChE adduct is a three-step process, while the reaction of hydroxylamine anion with the VX-AChE adduct seems to be a two-step process. The rate-determining step in the process is the initial attack on the VX of the VX-AChE adduct by the nucleophile. The subsequent steps are exergonic in nature. The potential energy surface (PES) for the reaction of the VX-AChE adduct with hydroxylamine anion reveals that the reactivation process is facilitated by the lower free energy of activation (by a factor of 1.7 kcal mol(-1)) than that of the formoximate anion at the B3LYP/6-311 G(d,p) level of theory. The higher free energy of activation for the reverse reactivation reaction between hydroxylamine anion and the VX-serine adduct further suggests that the hydroxylamine anion is a very good antidote agent for the reactivation process. The activation barriers calculated in solvent using the polarizable continuum model (PCM) for the reactivation of the VX-AChE adduct with hydroxylamine anion were also found to be low. The calculated results suggest that V-series compounds can be more toxic than G-series compounds, which is in accord with earlier experimental observations.

The lignicolous fungus Trametes versicolor (L.) Lloyd (1920): a promising natural source of antiradical and AChE inhibitory agents.

PubMed

Janjušević, Ljiljana; Karaman, Maja; Šibul, Filip; Tommonaro, Giuseppina; Iodice, Carmine; Jakovljević, Dragica; Pejin, Boris

2017-12-01

This study aimed to determine antiradical (DPPH • and • OH) and acetylcholinesterase (AChE) inhibitory activities along with chemical composition of autochtonous fungal species Trametes versicolor (Serbia). A total of 38 phenolic compounds with notable presence of phenolic acids were identified using HPLC/MS-MS. Its water extract exhibited the highest antiradical activity against • OH (3.21 μg/mL), among the rest due to the presence of gallic, p-coumaric and caffeic acids. At the concentration of 100 μg/mL, the same extract displayed a profound AChE inhibitory activity (60.53%) in liquid, compared to donepezil (89.05%), a drug in clinical practice used as positive control. The flavonoids baicalein and quercetin may be responsible compounds for the AChE inhibitory activity observed. These findings have demonstrated considerable potential of T. versicolor water extract as a natural source of antioxidant(s) and/or AChE inhibitor(s) to be eventually used as drug-like compounds or food supplements in the treatment of Alzheimer's disease.

A mid-infrared flow-through sensor for label-free monitoring of enzyme inhibition.

PubMed

Armenta, S; Tomischko, W; Lendl, B

2008-12-01

Label-free monitoring of acetylcholinesterase (AChE) activity was achieved with a mid-infrared flow-through sensor. The flow-through sensor comprised agarose beads, carrying covalently immobilized AChE, which were placed in a temperature-controlled (37 degrees C) CaF(2) flow cell with an optical path of 60 mum. The sensor was incorporated into a computer-controlled sequential injection (SI) system for automated liquid handling. Different mixtures of enzyme substrate acetylcholine (ACh) and inhibitor (tacrine) were prepared and fed into the flow-through sensor. The flow was stopped as soon as the prepared mixtures reached the sensor. Enzymatic hydrolysis of ACh by AChE was directly monitored as it took place in the flow-through sensor. The inhibition effect of tacrine was calculated from the reaction-induced spectral changes, revealing an important decrease in the activity of AChE, approaching zero when the inhibitor concentration is high enough. The developed mid-infrared flow-through sensor is flexible and can be used to study the inhibitor activity of different target molecules as well as different enzymes.

Effect of single and binary combinations of plant-derived molluscicides on different enzyme activities in the nervous tissue of Achatina fulica.

PubMed

Rao, I G; Singh, Amrita; Singh, V K; Singh, D K

2003-01-01

Effect of single and binary treatments of plant-derived molluscicides on different enzymes--acetylcholinesterase (AChE), lactic dehydrogenase (LDH) and acid/alkaline phosphatase (ACP/ALP)--in the nervous tissue of the harmful terrestrial snail Achatina fulica were studied. Sublethal in vivo 24-h exposure to 40% and 80% LC(50) of Azadirachta indica oil, Cedrus deodara oil, Allium sativum bulb powder, Nerium indicum bark powder and binary combinations of A. sativum (AS) + C. deodara (CD) and CD + A. indica (AI) oils significantly altered the activity of these enzymes in the nervous tissue of Achatina fulica. The binary treatment of AS + CD was more effective against AChE, LDH, and ALP than the single ones. However, binary treatment of AI + CD was more effective against ALP. Copyright 2003 John Wiley & Sons, Ltd.

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

Synthetic Helizyme Enzymes.

DTIC Science & Technology

1987-08-18

NOTATION 17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number) FIELD GROUP SUB-GROUP I Synthetic enzymes...chymotrypsin; molecular modeling; 03 peptide synthesis 19. ABSTRACT (Continue on reverse if necessary and identify by block number) The object of this...for AChE. Additionally, synthetic models ofcL- chymotrypsin built using cyclo- dextrins show catalytic activity over a limited pH range.2 Using L

Effect of chlorpyrifos and enrofloxacin on selected enzymes in rats.

PubMed

Barski, D; Spodniewska, A

2018-03-01

This study examined the effect of chlorpyrifos and/or enrofloxacin on the activity of acetylcholinesterase (AChE) in the blood and brain, and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. The experiment was conducted on Wistar strain rats. Chlorpyrifos was administered with a stomach tube at a dose of 0.04 LD50 for 28 days and enrofloxacin at a dose of 5 mg/kg bw for 5 consecutive days. The experiment found that enrofloxacin changed the activity of the enzymes under study only to a small extent. At the dose applied in the experiment, chlorpyrifos decreased the activity of AChE significantly, both in blood and in the brain, and increased the activity of ALT and AST in rat serum. The administration of chlorpyrifos in combination with enrofloxacin changed the activity of the enzymes under study only slightly. A weaker, but longer, inhibition of AChE activity in both blood and the brain was observed in this group compared to the animals exposed only to chlorpyrifos. However, although enrofloxacin, like chlorpyrifos, increases the activity of ALT and AST in serum, their combined administration did not increase the hepatotoxic effect. Copyright© by the Polish Academy of Sciences.

One-pot microwave assisted stereoselective synthesis of novel dihydro-2'H-spiro[indene-2,1'-pyrrolo-[3,4-c]pyrrole]-tetraones and evaluation of their antimycobacterial activity and inhibition of AChE.

PubMed

Bharkavi, Chelliah; Vivek Kumar, Sundaravel; Ashraf Ali, Mohamed; Osman, Hasnah; Muthusubramanian, Shanmugam; Perumal, Subbu

2017-07-15

An efficient one-pot microwave assisted stereoselective synthesis of novel dihydro-2'H-spiro[indene-2,1'-pyrrolo[3,4-c]pyrrole]-tetraone derivatives through three-component 1,3-dipolar cycloaddition of azomethine ylides generated in situ from ninhydrin and sarcosine with a series of 1-aryl-1H-pyrrole-2,5-diones is described. The synthesised compounds were screened for their antimycobacterial and AChE inhibition activities. Compound 4b (IC 50 1.30µM) has been found to display twelve fold antimycobacterial activity compared to cycloserine and it is thirty seven times more active than pyrimethamine. Compound 4h displays maximum AchE inhibitory activity with IC 50 value of 0.78±0.01µmol/L. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of EGCG and Chlorpyrifos on the Mortality, AChE and GSH of Adult Zebrafish: Independent and Combination

NASA Astrophysics Data System (ADS)

Zhang, Rong; Zhang, Jian; Gao, Qian; Guo, Nichun

2018-01-01

Chlorpyrifos is a neurotoxic agent and also causes oxidative stress in the body. EGCG is a typical strong antioxidant and has been reported to be neuroprotective. Our study investigated the mortality, the activity of acetylcholinesterase (AChE) in the brain and glutathione (GSH) in the liver of the adult Zebrafish in present of Chlorpyrifos and EGCG independent and combination. The results indicated that after the addition of EGCG, the mortality of zebrafish induced by Chlorpyrifos was reduced and the activity of AChE and glutathione (GSH) inhibited by Chlorpyrifos in zebrafish was significantly increased, which demonstrated that EGCG inhibited the toxicity Chlorpyrifos to zebrafish. The inhibition was dependent on the concentration of EGCG and Chlorpyrifos, which was not shown a gradual change trend but a complex situation.

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

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

Abstract 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

Neurotoxicity fingerprinting of venoms using on-line microfluidic AChBP profiling.

PubMed

Slagboom, Julien; Otvos, Reka A; Cardoso, Fernanda C; Iyer, Janaki; Visser, Jeroen C; van Doodewaerd, Bjorn R; McCleary, Ryan J R; Niessen, Wilfried M A; Somsen, Govert W; Lewis, Richard J; Kini, R Manjunatha; Smit, August B; Casewell, Nicholas R; Kool, Jeroen

2018-06-15

Venoms from snakes are rich sources of highly active proteins with potent affinity towards a variety of enzymes and receptors. Of the many distinct toxicities caused by envenomation, neurotoxicity plays an important role in the paralysis of prey by snakes as well as by venomous sea snails and insects. In order to improve the analytical discovery component of venom toxicity profiling, this paper describes the implementation of microfluidic high-resolution screening (HRS) to obtain neurotoxicity fingerprints from venoms that facilitates identification of the neurotoxic components of envenomation. To demonstrate this workflow, 47 snake venoms were profiled using the acetylcholine binding protein (AChBP) to mimic the target of neurotoxic proteins, in particular nicotinic acetylcholine receptors (nAChRs). In the microfluidic HRS system, nanoliquid chromatographic (nanoLC) separations were on-line connected to both AChBP profiling and parallel mass spectrometry (MS). For virtually all neurotoxic elapid snake venoms tested, we obtained bioactivity fingerprints showing major and minor bioactive zones containing masses consistent with three-finger toxins (3FTxs), whereas, viperid and colubrid venoms showed little or no detectable bioactivity. Our findings demonstrate that venom interactions with AChBP correlate with the severity of neurotoxicity observed following human envenoming by different snake species. We further, as proof of principle, characterized bioactive venom peptides from a viperid (Daboia russelli) and an elapid (Aspidelaps scutatus scutatus) snake by nanoLC-MS/MS, revealing that different toxin classes interact with the AChBP, and that this binding correlates with the inhibition of α7-nAChR in calcium-flux cell-based assays. The on-line post-column binding assay and subsequent toxin characterization methodologies described here provide a new in vitro analytic platform for rapidly investigating neurotoxic snake venom proteins. Copyright © 2018 The Author

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Wei; NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Shanghai 200032; Li, Juan

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 55.3more » 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.« less

Discovery of potent and selective acetylcholinesterase (AChE) inhibitors: acacetin 7-O-methyl ether Mannich base derivatives synthesised from easy access natural product naringin.

PubMed

Liu, Hao-Ran; Men, Xue; Gao, Xiao-Hui; Liu, Lin-Bo; Fan, Hao-Qun; Xia, Xin-Hua; Wang, Qiu-An

2018-03-01

Naringin, as a component universal existing in the peel of some fruits or medicinal plants, was usually selected as the material to synthesise bioactive derivates since it was easy to gain with low cost. In present investigation, eight new acacetin-7-O-methyl ether Mannich base derivatives (1-8) were synthesised from naringin. The bioactivity evaluation revealed that most of them exhibited moderate or potent acetylcholinesterase (AChE) inhibitory activity. Among them, compound 7 (IC 50 for AChE = 0.82 ± 0.08 μmol•L -1 , IC 50 for BuChE = 46.30 ± 3.26 μmol•L -1 ) showed a potent activity and high selectivity compared with the positive control Rivastigmine (IC 50 for AChE = 10.54 ± 0.86 μmol•L -1 , IC 50 for BuChE = 0.26 ± 0.08 μmol•L -1 ). The kinetic study suggested that compound 7 bind to AChE with mix-type inhibitory profile. Molecular docking study revealed that compound 7 could combine both catalytic active site (CAS) and peripheral active site (PAS) of AChE with four points (Trp84, Trp279, Tyr70 and Phe330), while it could bind with BuChE via only His 20.

Acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase, lactate dehydrogenase and catalase of the mosquitofish, Gambusia holbrooki.

PubMed

Nunes, B; Carvalho, F; Guilhermino, L

2004-12-01

The objective of this study was to investigate both acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and catalase (CAT) of the mosquitofish (Gambusia holbrooki). AChE, commonly used as a biomarker of neurotoxicity, was determined in the total head. LDH, an important enzyme of anaerobic metabolism, was quantified in dorsal muscle, and CAT, enzyme which has been used as indicative parameter of peroxisome proliferation, was determined in the liver. Furthermore, alterations of body and liver weight were also determined, through the calculation of the ratios final body weight/initial body weight, liver weight/final body weight, liver weight/gills weight and liver weight/head weight. Acute exposure of G. holbrooki to both clofibrate and clofibric acid induced a decrease in liver CAT activity, an increase in muscle LDH activity, while no effects were observed on AChE activity. However, chronic exposure did not alter significantly the enzymatic activities, suggesting reduced or null effects over these pathways, relative to effects reported in other species. No effects were observed for the calculated ratios, except a significant weight reduction for males chronically exposed to clofibrate.

Concomitant alpha7 and beta2 nicotinic AChR subunit deficiency leads to impaired energy homeostasis and increased physical activity in mice.

PubMed

Somm, Emmanuel; Guérardel, Audrey; Maouche, Kamel; Toulotte, Audrey; Veyrat-Durebex, Christelle; Rohner-Jeanrenaud, Françoise; Maskos, Uwe; Hüppi, Petra S; Schwitzgebel, Valérie M

2014-05-01

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels well characterized in neuronal signal transmission. Moreover, recent studies have revealed nAChR expression in nonneuronal cell types throughout the body, including tissues involved in metabolism. In the present study, we screen gene expression of nAChR subunits in pancreatic islets and adipose tissues. Mice pancreatic islets present predominant expression of α7 and β2 nAChR subunits but at a lower level than in central structures. Characterization of glucose and energy homeostasis in α7β2nAChR(-/-) mice revealed no major defect in insulin secretion and sensitivity but decreased glycemia apparently unrelated to gluconeogenesis or glycogenolysis. α7β2nAChR(-/-) mice presented an increase in lean and bone body mass and a decrease in fat storage with normal body weight. These observations were associated with elevated spontaneous physical activity in α7β2nAChR(-/-) mice, mainly due to elevation in fine vertical (rearing) activity while their horizontal (ambulatory) activity remained unchanged. In contrast to α7nAChR(-/-) mice presenting glucose intolerance and insulin resistance associated to excessive inflammation of adipose tissue, the present metabolic phenotyping of α7β2nAChR(-/-) mice revealed a metabolic improvement possibly linked to the increase in spontaneous physical activity related to central β2nAChR deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

Reciprocal activation of α5-nAChR and STAT3 in nicotine-induced human lung cancer cell proliferation.

PubMed

Zhang, Yao; Jia, Yanfei; Li, Ping; Li, Huanjie; Xiao, Dongjie; Wang, Yunshan; Ma, Xiaoli

2017-07-20

Cigarette smoking is the top environmental risk factor for lung cancer. Nicotine, the addictive component of cigarettes, induces lung cancer cell proliferation, invasion and migration via the activation of nicotinic acetylcholine receptors (nAChRs). Genome-wide association studies (GWAS) show that CHRNA5 gene encoding α5-nAChR is especially relevant to lung cancer. However, the mechanism of this subunit in lung cancer is not clear. In the present study, we demonstrate that the expression of α5-nAChR is correlated with phosphorylated STAT3 (pSTAT3) expression, smoking history and lower survival of non-small cell lung cancer (NSCLC) samples. Nicotine increased the levels of α5-nAChR mRNA and protein in NSCLC cell lines and activated the JAK2/STAT3 signaling cascade. Nicotine-induced activation of JAK2/STAT3 signaling was inhibited by the silencing of α5-nAChR. Characterization of the CHRNA5 promoter revealed four STAT3-response elements. ChIP assays confirmed that the CHRNA5 promoter contains STAT3 binding sites. By silencing STAT3 expression, nicotine-induced upregulation of α5-nAChR was suppressed. Downregulation of α5-nAChR and/or STAT3 expression inhibited nicotine-induced lung cancer cell proliferation. These results suggest that there is a feedback loop between α5-nAChR and STAT3 that contributes to the nicotine-induced tumor cell proliferation, which indicates that α5-nAChR is an important therapeutic target involved in tobacco-associated lung carcinogenesis. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Acetylcholinesterase (AChE) and heat shock proteins (Hsp70) of gypsy moth (Lymantria dispar L.) larvae in response to long-term fluoranthene exposure.

PubMed

Mrdaković, Marija; Ilijin, Larisa; Vlahović, Milena; Matić, Dragana; Gavrilović, Anja; Mrkonja, Aleksandra; Perić-Mataruga, Vesna

2016-09-01

Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in living organisms, thus impairing fitness related traits and influencing their populations. This imposes the need for providing early-warning signals of pollution. Our study aimed to examine changes in the activity of acetylcholinesterase (AChE) and the concentration of heat shock proteins (Hsp70) in homogenates of brain tissues of fifth instar gypsy moth (Lymantria dispar L.) larvae, exposed to the ubiquitous PAH, fluoranthene, supplemented to the rearing diet. Significantly increased activity of AChE in larvae fed on the diets with high fluoranthene concentrations suggests the necessity for elucidation of the role of AChE in these insects when exposed to PAH pollution. Significant induction of Hsp70 in gypsy moth larvae reared on the diets containing low fluoranthene concentrations, indicate that changes in the level of Hsp70 might be useful as an indicator of pollution in this widespread forest species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of hexamethonium, phenothiazines, propranolol and ephedrine on acetylcholinesterase carbamylation by physostigmine, aldicarb and carbaryl: interaction between the active site and the functionally distinct peripheral sites in acetylcholinesterase.

PubMed

Singh, A K; Spassova, D

1998-01-01

Physostigmine, aldicarb and carbaryl were potent inhibitors of acetylcholinesterase (AChE). The physostigmine-inhibited AChE fluoresced at 300 nm excitation and 500 nm emission wavelengths, but the aldicarb and carbaryl inhibited enzyme did not. This suggests that the carbamylated active center is not the fluorescing site in AChE. The fluorescence intensity of physostigmine-inhibited AChE decreased with increasing the substrate (acetylthiocholine) concentration, thus indicating that physostigmine binding to the active site is essential for the development of fluorescence. Thus, the physostigmine-inhibited AChE fluoresces due to the binding of trimethylpyrrolo[2,3-b]indol (TMPI) moiety, formed by the hydrolysis of physostigmine, to a peripheral site in AChE. The fluorescence intensity of the physostigmine-inhibited enzyme decreased when the inhibited-enzyme was dialyzed for either 30 min that poorly reactivated the enzyme or 180 min that fully reactivated the enzyme. This suggests that dialysis dissociates the AChE-TMPI complex much faster than it reactivates the carbamylated AChE. Ephedrine, propranolol and phenothiazines including trifluoparazine (TPZ) caused non-competitive inhibition, while hexamethonium caused an uncompetitive inhibition of AChE activity. TPZ, upon binding with AChE, formed a fluorescent TPZ-enzyme complex. The fluorescence intensity of TPZ-AChE complex was effectively decreased by ephedrine, but not by propranolol or hexamethonium. This indicates that TPZ and ephedrine bind to the same site in AChE which is different from the site/or sites to which propranolol or hexamethonium bind. Hexamethonium protected AChE from inhibition by carbamates and decreased the fluorescence intensity of the physostigmine-inhibited AChE. Phenothiazines and ephedrine did not modulate the enzyme inhibition or the fluorescence intensity of the physostigmine-inhibited AChE. Propranolol and TPZ potentiated the enzyme inhibition and increased the fluorescence intensity

Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT Assay and Larvicidal Activities.

PubMed

Tavakoli, Saeed; Vatandoost, Hassan; Zeidabadinezhad, Reza; Hajiaghaee, Reza; Hadjiakhoondi, Abbas; Abai, Mohammad Reza; Yassa, Narguess

2017-09-01

We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant. Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Antioxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anopheles stephensi was carried out according to the method described by WHO. In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-verbenol (9.66%), isobutyl acetate (25.73%) and E-β-caryophyllene (8.68%) were main compounds. The oil showed (IC 50 = 111.2μg/ml) in DPPH and IC 50 = 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD 50 = 1.1μg/ml) in brine shrimp lethality test and with (IC 50 = 22.0, 25.0 and 42.55 μg/ml) on three cancerous cell lines (MCF-7, A-549 and HT-29) respectively. LC 50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli , Aspergillus niger and Candida albicans. The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines.

Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT Assay and Larvicidal Activities

PubMed Central

Tavakoli, Saeed; Vatandoost, Hassan; Zeidabadinezhad, Reza; Hajiaghaee, Reza; Hadjiakhoondi, Abbas; Abai, Mohammad Reza; Yassa, Narguess

2017-01-01

Background: We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant. Methods: Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Antioxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anopheles stephensi was carried out according to the method described by WHO. Results: In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-verbenol (9.66%), isobutyl acetate (25.73%) and E-β-caryophyllene (8.68%) were main compounds. The oil showed (IC50= 111.2μg/ml) in DPPH and IC50= 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD50= 1.1μg/ml) in brine shrimp lethality test and with (IC50= 22.0, 25.0 and 42.55 μg/ml) on three cancerous cell lines (MCF-7, A-549 and HT-29) respectively. LC50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli, Aspergillus niger and Candida albicans. Conclusion: The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines. PMID:29322058

Antioxidant, Acetylcholinesterase, Butyrylcholinesterase, and α-glucosidase Inhibitory Activities of Corchorus depressus

PubMed Central

Afzal, Samina; Chaudhry, Bashir Ahmad; Ahmad, Ashfaq; Uzair, Muhammad; Afzal, Khurram

2017-01-01

Background: Corchorus depressus (Cd) commonly known as Boa-phalee belonging to the family Tiliaceae having 50 genera and 450 species. Cd is not among the studied medicinal agent despite its potential in ethnopharmacology. Objectives: The present study investigated antioxidant, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase inhibitory activities of Cd. The dichloromethane and methanolic extracts of the Cd were evaluated for biological activities such as antioxidant and enzyme inhibitory activities of AChE, BChE, and α-glucosidase. Materials and Methods: Antioxidant activity was evaluated by measuring free radical scavenging potential of Cd using 1,1-diphenyl-2-picrylhydrazyl. Enzyme inhibition activities were done by measuring optical density. Results: The methanol extract of roots of Cd showed potential free radical scavenging activity 99% at concentration 16.1 μg/ml. AChE was inhibited by aerial part of dichloromethane fraction by 46.07% ± 0.45% while dichloromethane extracts of roots of Cd possessed significant activity against BChE with 86% inhibition compared with standard drug Eserine at concentration 0.5 mg/ml. The dichloromethane extract of roots of Cd showed 79% inhibition against α-glucosidase enzyme activity with IC50 62.8 ± 1.5 μg/ml. Conclusion: These findings suggest Cd as useful therapeutic option as antioxidant and inhibition of AChE, BChE, and α-glucosidase activities. SUMMARY The aerial parts and roots of Corchorus depressus (Cd) were extracted in dichloromethane and methanolThe extract of roots of Cd showed free radical scavenging activity 99% at concentration 16.1 mg/ml, Ach inhibition by aerial parts of dichloromethane fraction by 46.07%, and 79% inhibition against a-glucosidase enzyme activity with IC50 62.8 ± 1.5 mg/mlThe dichloromethane and methanolic extracts of Cd exhibited antioxidant inhibition of acetyl cholinesterase, butyrylcholinesterase, and a-glucosidase activities. Abbreviations used: DPPH: 1

Cholinesterase enzymes inhibitors from the leaves of Rauvolfia reflexa and their molecular docking study.

PubMed

Fadaeinasab, Mehran; Hadi, A Hamid A; Kia, Yalda; Basiri, Alireza; Murugaiyah, Vikneswaran

2013-03-25

Plants of the Apocynaceae family have been traditionally used in the treatment of age-related brain disorders. Rauvolfia reflexa, a member of the family, has been used as an antidote for poisons and to treat malaria. The dichloromethane, ethanol and methanol extracts from the leaves of Rauvolfia reflexa showed potential acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, with IC50 values in the 8.49 to 52.23 g/mL range. Further cholinesterase inhibitory-guided isolation of these extracts afforded four bioactive compounds, namely: (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (1), (E)-methyl 3-(4-hydroxy-3,5-dimethoxyphenyl) acrylate (2), 17-methoxycarbonyl-14-heptadecaenyl-4-hydroxy-3-methoxycinnamate (3) and 1,2,3,4-tetrahydro-1-oxo-β-carboline (4). The isolated compounds showed moderate cholinesterase inhibitory activity compared to the reference standard, physostigmine. Compounds 1 and 2 showed the highest inhibitory activity against AChE (IC50 = 60.17 µM) and BChE (IC50 = 61.72 µM), respectively. Despite having similar molecular weight, compounds 1 and 2 were structurally different according to their chemical substitution patterns, leading to their different enzyme inhibition selectivity. Compound 2 was more selective against BChE, whereas compound 1 was a selective inhibitor of AChE. Molecular docking revealed that both compounds 1 and 2 were inserted, but not deeply into the active site of the cholinesterase enzymes.

ACh-induced endothelial NO synthase translocation, NO release and vasodilatation in the hamster microcirculation in vivo

PubMed Central

Figueroa, Xavier F; González, Daniel R; Martínez, Agustín D; Durán, Walter N; Boric, Mauricio P

2002-01-01

Studies in cultured cells show that activation of endothelial nitric oxide (NO) synthase (eNOS) requires the dissociation of this enzyme from its inhibitory association with caveolin-1 (Cav-1), and perhaps its translocation from plasma membrane caveolae to other cellular compartments. We investigated the hypothesis that in vivo NO-dependent vasodilatation is associated with the translocation of eNOS from the cell membrane. To this end, we applied ACh topically (10-100 μm for 10 min) to the hamster cheek pouch microcirculation and measured NO production, blood flow and vessel diameter, and assessed subcellular eNOS distribution by Western blotting. Baseline NO production was 54.4 ± 5.2 pmol min−1 (n = 16). ACh increased NO release, caused arteriolar and venular dilatation and elevated microvascular flow. These responses were inhibited by NG-nitro-L-arginine (30 μm). The maximal increase in NO production induced by 10 μm and 100 μm ACh was 45 ± 20 % and 111 ± 33 %, respectively; the corresponding blood flow increases were 50 ± 10 % and 130 ± 24 %, respectively (n = 4-6). Both responses followed a similar time course, although increases in NO preceded flow changes. In non-stimulated tissues, eNOS was distributed mainly in the microsomal fraction. ACh-induced vasodilatation was associated with eNOS translocation to the cytosolic and Golgi-enriched fractions. After 1.5, 3.0 or 6.0 min of application, 10 μm ACh decreased the level of membrane-bound eNOS by -13 ± 4 %, -60 ± 4 % and -19 ± 17 %, respectively; at the same time points, 100 μm ACh reduced microsomal eNOS content by -38 ± 9 %, -61 ± 16 % and -40 ± 18 %, respectively (n = 4-5). In all cases, microsomal Cav-1 content did not change. The close ACh concentration dependence and the concomitance between eNOS subcellular redistribution and NO release support the concept that eNOS translocation from the plasma membrane is part of an activation mechanism that induces NO-dependent vasodilatation in

Behavior of detoxifying enzymes of Aedes aegypti exposed to girgensohnine alkaloid analog and Cymbopogon flexuosus essential oil.

PubMed

Carreño Otero, Aurora L; Palacio-Cortés, Angela Maria; Navarro-Silva, Mario Antonio; Kouznetsov, Vladimir V; Duque L, Jonny E

2018-01-01

Because mosquito control depend on the use of commercial insecticides and resistance has been described in some of them, there is a need to explore new molecules no resistant. In vivo effects of girgensohnine analog 2-(3,4-dimethoxyphenyl)-2-(piperidin-1-yl)acetonitrile DPPA and Cymbopogon flexuosus essential oil CFEO, on the detoxifying enzymes acetylcholinesterase (AChE), glutathione-S-transferase (GST), nonspecific esterases (α- and β-), mixed function oxidases (MFO) and p-NPA esterases were evaluated on a Rockefeller (Rock) and wild Aedes aegypti population from Santander, Colombia (WSant). The action was tested after 24h of exposure at concentrations of 20.10, 35.18 and 70.35mgL -1 of DPPA and 18.45, 30.75 and 61.50mgL -1 of CFEO, respectively. It was found that AChE activity of Rock and WSant was not influenced by the evaluated concentration of DPPA and CFEO (p>0.05), while MFO activity was significantly affected by all CFEO concentrations in WSant (p<0.05). GST, α- and β-esterase activities were affected in Rock exposed at the highest CFEO concentration, this concentration also modified β-esterases activity of WSant. DPPA and CFEO sublethal doses induced inhibition of AChE activity on untreated larvae homogenate from 12 to 20% and 18 to 26%, respectively. For untreated adult homogenate, the inhibition activity raised up to 14 to 27% for DPPA and 26 to 34% for CFEO. Elevated levels of detoxifying enzymes, found when CFEO was evaluated, showed a larval sensitivity not observed by the pure compound suggesting that DPPA, contrary to CFEO, was not recognized, transformed or eliminated by the evaluated detoxifying enzymes. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of hyper- and hypothyroidism on acetylcholinesterase, (Na(+), K (+))- and Mg ( 2+ )-ATPase activities of adult rat hypothalamus and cerebellum.

PubMed

Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Stolakis, Vasileios; Mourouzis, Iordanis; Cokkinos, Dennis; Tsakiris, Stylianos

2007-03-01

Thyroid hormones (THs) are recognized as key metabolic hormones, and the metabolic rate increases in hyperthyroidism, while it decreases in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na(+), K(+))- and Mg(2+)-ATPase in the hypothalamus and the cerebellum of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. Neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE (-23%, p < 0.001) and the Na(+), K(+)-ATPase activities (-26%, p < 0.001). Moreover, hypothyroidism had a similar effect on the examined enzyme activities: AChE (-17%, p < 0.001) and Na(+), K(+)-ATPase (-27%, p < 0.001). Mg(2+)-ATPase activity was found unaltered in both the hyper- and the hypothyroid brain regions. neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE and the Na(+), K(+)-ATPase activities. The decreased (by the THs) Na(+), K(+)-ATPase activities may increase the synaptic acetylcholine release, and thus, could result in a decrease in the cerebellar AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems.

The role of the peripheral anionic site and cation-pi interactions in the ligand penetration of the human AChE gorge.

PubMed

Branduardi, Davide; Gervasio, Francesco Luigi; Cavalli, Andrea; Recanatini, Maurizio; Parrinello, Michele

2005-06-29

We study the ligand (tetramethylammonium) recognition by the peripheral anionic site and its penetration of the human AChE gorge by using atomistic molecular dynamics simulations and our recently developed metadynamics method. The role of both the peripheral anionic site and the formation of cation-pi interactions in the ligand entrance are clearly shown. In particular, a simulation with the W286A mutant shows the fundamental role of this residue in anchoring the ligand at the peripheral anionic site of the enzyme and in positioning it prior to the gorge entrance. Once the ligand is properly oriented, the formation of specific and synchronized cation-pi interactions with W86, F295, and Y341 enables the gorge penetration. Eventually, the ligand is stabilized in a free energy basin by means of cation-pi interactions with W86.

Effects of experimentally-induced maternal hypothyroidism on crucial offspring rat brain enzyme activities.

PubMed

Koromilas, Christos; Liapi, Charis; Zarros, Apostolos; Stolakis, Vasileios; Tsagianni, Anastasia; Skandali, Nikolina; Al-Humadi, Hussam; Tsakiris, Stylianos

2014-06-01

Hypothyroidism is known to exert significant structural and functional changes to the developing central nervous system, and can lead to the establishment of serious mental retardation and neurological problems. The aim of the present study was to shed more light on the effects of gestational and/or lactational maternal exposure to propylthiouracil-induced experimental hypothyroidism on crucial brain enzyme activities of Wistar rat offspring, at two time-points of their lives: at birth (day-1) and at 21 days of age (end of lactation). Under all studied experimental conditions, offspring brain acetylcholinesterase (AChE) activity was found to be significantly decreased due to maternal hypothyroidism, in contrast to the two studied adenosinetriphosphatase (Na(+),K(+)-ATPase and Mg(2+)-ATPase) activities that were only found to be significantly altered right after birth (increased and decreased, respectively, following an exposure to gestational maternal hypothyroidism) and were restored to control levels by the end of lactation. As our findings regarding the pattern of effects that maternal hypothyroidism has on the above-mentioned crucial offspring brain enzyme activities are compared to those reported in the literature, several differences are revealed that could be attributed to both the mode of the experimental simulation approach followed as well as to the time-frames examined. These findings could provide the basis for a debate on the need of a more consistent experimental approach to hypothyroidism during neurodevelopment as well as for a further evaluation of the herein presented and discussed neurochemical (and, ultimately, neurodevelopmental) effects of experimentally-induced maternal hypothyroidism, in a brain region-specific manner. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward.

PubMed

Henderson, Brandon J; Wall, Teagan R; Henley, Beverley M; Kim, Charlene H; Nichols, Weston A; Moaddel, Ruin; Xiao, Cheng; Lester, Henry A

2016-03-09

Upregulation of β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate β2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(β2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(β2)3 nAChRs. Menthol alone also increases the number of α6β2 receptors that exclude the β3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway. Copyright © 2016 the authors 0270-6474/16/362957-18$15.00/0.

Photolabeling a Nicotinic Acetylcholine Receptor (nAChR) with an (α4)3(β2)2 nAChR-Selective Positive Allosteric Modulator

PubMed Central

Deba, Farah; Wang, Ze-Jun; Cohen, Jonathan B.

2016-01-01

Positive allosteric modulators (PAMs) of nicotinic acetylcholine (ACh) receptors (nAChRs) have potential clinical applications in the treatment of nicotine dependence and many neuropsychiatric conditions associated with decreased brain cholinergic activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM selective for neuronal nAChRs containing the α4 subunit. In this report, we compare CMPI interactions with low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs, and with muscle-type nAChRs. In addition, we use the intrinsic reactivity of [3H]CMPI upon photolysis at 312 nm to identify its binding sites in Torpedo nAChRs. Recording from Xenopus oocytes, we found that CMPI potentiated maximally the responses of (α4)3(β2)2 nAChR to 10 μM ACh (EC10) by 400% and with an EC50 of ∼1 µM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10 µM) ACh responses of (α4)2(β2)3 nAChRs and fully inhibited human muscle and Torpedo nAChRs with IC50 values of ∼0.5 µM. Upon irradiation at 312 nm, [3H]CMPI photoincorporated into each Torpedo [(α1)2β1γδ] nAChR subunit. Sequencing of peptide fragments isolated from [3H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr190, αTyr198, γTrp55, γTyr111, γTyr117, δTrp57) that was fully inhibitable by agonist and lower-efficiency, state-dependent [3H]CMPI photolabeling within the ion channel. Our results establish that CMPI is a potent potentiator of nAChRs containing an α4:α4 subunit interface, and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2 nAChR. PMID:26976945

Antioxidant and cholinesterase inhibitory activity of a new peptide from Ziziphus jujuba fruits.

PubMed

Zare-Zardini, Hadi; Tolueinia, Behnaz; Hashemi, Azam; Ebrahimi, Leila; Fesahat, Farzaneh

2013-11-01

Antioxidant agents and cholinesterase inhibitors are the foremost drugs for the treatment of Alzheimer's disease (AD). In this study, a new peptide from Ziziphus jujuba fruits was investigated for its inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes as well as antioxidant activity. This peptide was introduced as a new peptide and named Snakin-Z. The Snakin-Z displayed considerable cholinesterase inhibition against AChE and BChE. The half maximal inhibitory concentration (IC50) values of Snakin-Z against AChE and BChE are 0.58 ± 0.08 and 0.72 ± 0.085 mg/mL, respectively. This peptide has 80% enzyme inhibitory activity on AChE and BChE at 1.5 mg/mL. The Snakin-Z also had the high antioxidant activity (IC50 = 0.75 ± 0.09 mg/mL). Thus, it is suggested that Snakin-Z may be beneficial in the treatment of AD. However, more detailed researches are still required as in vivo testing its anticholinesterase and antioxidant activities.

Synthesis, biological evaluation, and computational studies of Tri- and tetracyclic nitrogen-bridgehead compounds as potent dual-acting AChE inhibitors and hH3 receptor antagonists.

PubMed

Darras, Fouad H; Pockes, Steffen; Huang, Guozheng; Wehle, Sarah; Strasser, Andrea; Wittmann, Hans-Joachim; Nimczick, Martin; Sotriffer, Christoph A; Decker, Michael

2014-03-19

Combination of AChE inhibiting and histamine H3 receptor antagonizing properties in a single molecule might show synergistic effects to improve cognitive deficits in Alzheimer's disease, since both pharmacological actions are able to enhance cholinergic neurotransmission in the cortex. However, whereas AChE inhibitors prevent hydrolysis of acetylcholine also peripherally, histamine H3 antagonists will raise acetylcholine levels mostly in the brain due to predominant occurrence of the receptor in the central nervous system. In this work, we designed and synthesized two novel classes of tri- and tetracyclic nitrogen-bridgehead compounds acting as dual AChE inhibitors and histamine H3 antagonists by combining the nitrogen-bridgehead moiety of novel AChE inhibitors with a second N-basic fragment based on the piperidinylpropoxy pharmacophore with different spacer lengths. Intensive structure-activity relationships (SARs) with regard to both biological targets led to compound 41 which showed balanced affinities as hAChE inhibitor with IC50 = 33.9 nM, and hH3R antagonism with Ki = 76.2 nM with greater than 200-fold selectivity over the other histamine receptor subtypes. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds and molecular dynamics studies to explain high affinity at the hH3R.

Synthesis, characterization and cholinesterase enzymes inhibitory activity of 1-[3-methyl-5-(2,6,6-trimethyl-cyclohex-1-enyl)-4,5-dihydro-pyrazol-1-yl]-ethanone

NASA Astrophysics Data System (ADS)

Mehdi, Sayed Hasan; Ghalib, Raza Murad; Hashim, Rokiah; da Silva, M. Fátima C. Guedes; Sulaiman, Othman; Murugaiyah, Vikneswaran; Marimuthu, Mani Maran; Naqvi, Mehnaz

2013-10-01

The crystal structure of the title compound, 1-[3-methyl-5-(2,6,6-trimethyl-cyclohex-1-enyl)-4,5-dihydro-pyrazol-1-yl]-ethanone has been determined by single crystal X-ray diffraction. It crystallizes in the orthorhombic space group P212121. The FTIR as well as the 1H and 13C NMR spectra of the compound were also recorded and briefly discussed. Compound 1 demonstrated good inhibitory activity against butyrylcholinesterase (BChE; IC50 = 46.42 μM) comparable to physostigmine. However it showed moderate inhibitory activity against acetylcholinesterase (AChE; IC50 = 157.31 μM). It showed moderate inhibitory activity against acetylcholinesterase and selective inhibitory activity towards butyrylcholinesterase enzyme.

Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation.

PubMed

Hagstrom, Danielle; Hirokawa, Hideto; Zhang, Limin; Radic, Zoran; Taylor, Palmer; Collins, Eva-Maria S

2017-08-01

The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies.

Effects of argan oil on the mitochondrial function, antioxidant system and the activity of NADPH- generating enzymes in acrylamide treated rat brain.

PubMed

Aydın, Birsen

2017-03-01

Argan oil (AO) is rich in minor compounds such as polyphenols and tocopherols which are powerful antioxidants. Acrylamide (ACR) has been classified as a neurotoxic agent in animals and humans. Mitochondrial oxidative stress and dysfunction is one of the most probable molecular mechanisms of neurodegenerative diseases. Female Sprague Dawley rats were exposed to ACR (50mg/kg i.p. three times a week), AO (6ml/kg,o.p, per day) or together for 30days. The activities of cytosolic enzymes such as xanthine oxidase (XO), glucose 6-phosphate dehydrogenase (G6PDH), glutathione-S-transferase (GST), mitochondrial oxidative stress, oxidative phosphorylation (OXPHOS) and tricarboxylic acid cycle (TCA) enzymes, mitochondrial metabolic function, adenosine triphosphate (ATP) level and acetylcholinesterase (AChE) activity were assessed in rat brain. Cytosolic and mitochondrial antioxidant enzymes were significantly diminished in the brains of rats treated with ACR compared to those in control. Besides, ACR treatment resulted in a significant reduction in brain ATP level, mitochondrial metabolic function, OXPHOS and TCA enzymes. Administration of AO restored both the cytosolic and mitochondrial oxidative stress by normalizing nicotinamide adenine dinucleotide phosphate (NADPH) generating enzymes. In addition, improved mitochondrial function primarily enhancing nicotinamide adenine dinucleotide (NADH) generated enzymes activities and ATP level in the mitochondria. The reason for AO's obvious beneficial effects in this study may be due to synergistic effects of its different bioactive compounds which is especially effective on mitochondria. Modulation of the brain mitochondrial functions and antioxidant systems by AO may lead to the development of new mitochondria-targeted antioxidants in the future. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Cholinesterase inhibitors modify the activity of intrinsic cardiac neurons.

PubMed

Darvesh, Sultan; Arora, Rakesh C; Martin, Earl; Magee, David; Hopkins, David A; Armour, J Andrew

2004-08-01

Cholinesterase inhibitors used to treat the symptoms of Alzheimer's disease (AD) inhibit both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), albeit to different degrees. Because central and peripheral neurons, including intrinsic cardiac neurons located on the surface of the mammalian heart, express both BuChE and AChE, we studied spontaneously active intrinsic cardiac neurons in the pig as a model to assess the effects of inhibition of AChE compared to BuChE. Neuroanatomical experiments showed that some porcine intrinsic cardiac neurons expressed AChE and/or BuChE. Enzyme kinetic experiments with cholinesterase inhibitors, namely, donepezil, galantamine, (+/-) huperzine A, metrifonate, rivastigmine, and tetrahydroaminoacridine, demonstrated that these compounds differentially inhibited porcine AChE and BuChE. Donepezil and (+/-) huperzine A were better reversible inhibitors of AChE, and galantamine equally inhibited both the enzymes. Tetrahydroaminoacridine was a better reversible inhibitor of BuChE. Rivastigmine caused more rapid inactivation of BuChE as compared to AChE. Neurophysiological studies showed that acetylcholine and butyrylcholine increase or decrease the spontaneous activity of the intrinsic cardiac neurons. Donepezil, galantamine, (+/-) huperzine A, and tetrahydroaminoacridine changed spontaneous neuronal activity by about 30-35 impulses per minute, while rivastigmine changed it by approximately 100 impulses per minute. It is concluded that (i) inhibition of AChE and BuChE directly affects the porcine intrinsic cardiac nervous system, (ii) the intrinsic cardiac nervous system represents a suitable model for examining the effects of cholinesterase inhibitors on mammalian neurons in vivo, and (iii) the activity of intrinsic cardiac neurons may be affected by pharmacological agents that inhibit cholinesterases.

The Activity of Cholinesterases in Diapausing and Flying Red Mason Bees Osmia bicornis (Megachilidae).

PubMed

Dmochowska-Slezak, Kamila; Zaobidna, Ewa; Domeracka, Joanna; Swiatkowska, Marta; Rusznica, Małgorzata; Zółtowska, Krystyna

2015-01-01

The red mason bee (Osmia bicornis) is a highly effective pollinator that is exposed to various xenobiotics. The organism's potential resistance to the toxic effects of xenobiotics can be determined based on cholinesterase activity. The activity of cholinesterases (ChEs) towards acetylcholine (ACh) and butyrylcholine (BCh) was determined in extracts of diapausing (between October and late March) and flying bees (May). In both males and females, enzyme activity was higher towards ACh than towards BCh. The ratio of ACh/BCh activity was determined in the range of 1.43 to 4.15 in diapausing females and 3.00 to 7.18 in diapausing males. No significant changes in ChE activity towards ACh were observed in females before December and in males before February. Enzyme activity towards ACh increased dynamically in the second half of March. Enzyme activity towards BCh remained stable in both sexes until mid-March, after which it increased significantly. Excluding mid-March, enzyme BCh activity was significantly higher in females than in males. The activity of carboxylesterase towards 4-p-nitrophenyl butyrate was determined in females to assess the involvement of non-specific esterases in the hydrolysis of choline esters. Carboxylesterase activity was low in comparison with cholinesterase activity, and it remained practically unchanged throughout diapause, suggesting that choline esters in female O. bicornis extracts were hydrolyzed mainly by acetylcholinesterases.

Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward

PubMed Central

Henderson, Brandon J.; Wall, Teagan R.; Henley, Beverley M.; Kim, Charlene H.; Nichols, Weston A.; Moaddel, Ruin; Xiao, Cheng

2016-01-01

Upregulation of β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate β2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(β2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(β2)3 nAChRs. Menthol alone also increases the number of α6β2 receptors that exclude the β3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway. SIGNIFICANCE STATEMENT Menthol, the most popular flavorant for tobacco products, has been considered simply a benign flavor additive. However, as we show here

Inhibitory activities of major anthraquinones and other constituents from Cassia obtusifolia against β-secretase and cholinesterases.

PubMed

Jung, Hyun Ah; Ali, Md Yousof; Jung, Hee Jin; Jeong, Hyong Oh; Chung, Hae Young; Choi, Jae Sue

2016-09-15

Semen Cassiae has been traditionally used as an herbal remedy for liver, eye, and acute inflammatory diseases. Recent pharmacological reports have indicated that Cassiae semen has neuroprotective effects, attributable to its anti-inflammatory actions, in ischemic stroke and Alzheimer's disease (AD) models. The basic goal of this study was to evaluate the anti-AD activities of C. obtusifolia and its major constituents. Previously, the extract of C. obtusifolia seeds, was reported to have memory enhancing properties and anti-AD activity to ameliorate amyloid β-induced synaptic dysfunction. However, the responsible components of C. obtusifolia seeds in an AD are currently still unknown. In this study, we investigated the inhibitory effects of C. obtusifolia and its constituents against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) enzyme activity. In vitro cholinesterase enzyme assays by using AChE, BChE, and BACE1 were performed. We also scrutinized the potentials of Cassiae semen active component as BACE1 inhibitors via enzyme kinetics and molecular docking simulation. In vitro enzyme assays demonstrated that C. obtusifolia and its major constituents have promising inhibitory potential against AChE, BChE, and BACE1. All Cassiae semen constituents exhibited potent inhibitory activities against AChE and BACE1 with IC50 values of 6.29-109µg/mL and 0.94-190µg/mL, whereas alaternin, questin, and toralactone gentiobioside exhibited significant inhibitory activities against BChE with IC50 values of 113.10-137.74µg/mL. Kinetic study revealed that alaternin noncompetitively inhibited, whereas cassiaside and emodin showed mixed-type inhibition against BACE1. Furthermore, molecular docking simulation results demonstrated that hydroxyl group of alaternin and emodin tightly interacted with the active site residues of BACE1 and their relevant binding energies (-6.62 and -6.89kcal

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

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

PubMed

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.

Anti-Alzheimer's disease activity of compounds from the root bark of Morus alba L.

PubMed

Kuk, Eun Bi; Jo, A Ra; Oh, Seo In; Sohn, Hee Sook; Seong, Su Hui; Roy, Anupom; Choi, Jae Sue; Jung, Hyun Ah

2017-03-01

The inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays important roles in prevention and treatment of Alzheimer's disease (AD). Among the individual parts of Morus alba L. including root bark, branches, leaves, and fruits, the root bark showed the most potent enzyme inhibitory activities. Therefore, the aim of this study was to evaluate the anti-AD activity of the M. alba root bark and its isolate compounds, including mulberrofuran G (1), albanol B (2), and kuwanon G (3) via inhibition of AChE, BChE, and BACE1. Compounds 1 and 2 showed strong AChE- and BChE-inhibitory activities; 1-3 showed significant BACE1 inhibitory activity. Based on the kinetic study with AChE and BChE, 2 and 3 showed noncompetitive-type inhibition; 1 showed mixed-type inhibition. Moreover, 1-3 showed mixed-type inhibition against BACE1. The molecular docking simulations of 1-3 demonstrated negative binding energies, indicating a high affinity to AChE and BACE1. The hydroxyl group of 1-3 formed hydrogen bond with the amino acid residues located at AChE and BACE1. Consequently, these results indicate that the root bark of M. alba and its active compounds might be promising candidates for preventive and therapeutic agents for AD.

ACh-induced hyperpolarization and decreased resistance in mammalian type II vestibular hair cells.

PubMed

Poppi, Lauren A; Tabatabaee, Hessam; Drury, Hannah R; Jobling, Phillip; Callister, Robert J; Migliaccio, Americo A; Jordan, Paivi M; Holt, Joseph C; Rabbitt, Richard D; Lim, Rebecca; Brichta, Alan M

2018-01-01

In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9 -/- ) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9 -/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted

Novel Method of Preparation and Activity Research on Arctigenin from Fructus Arctii.

PubMed

Cai, Enbo; Han, Jiahong; Yang, Limin; Zhang, Weiyuan; Zhao, Yan; Chen, Qiulian; Guo, Meng; He, Xinhong

2018-01-01

Arctigenin has many pharmacological activities with clinical significance and is derived from Arctium lappa L. However, the present extraction method is inefficient and does not have meaningful industrial production. A new method to directly prepare arctigenin was established by combining enzyme-assisted extraction and central composite design. Arctigenin's further pharmacological activity was also surveyed in vitro . β-D-Glucosidase, a food-grade enzyme, was added directly to the fruits of A. lappa L. to hydrolyze the arctiin to arctigenin, and the obtained samples were subsequently subjected to ethanol (30%, v/v) extraction. The pharmacological activity of the extraction and arctigenin was determined by inhibiting acetylcholinesterase (AChE) and scavenging nitrite. The factors investigated include the enzyme concentration (0.5%-2.5%), ultrasound time (10 min -3 0 min), and extraction temperature (30°C-50°C). From the analysis of the results by Design-Expert (V8.0.6), the optimal extraction conditions were obtained: enzyme concentration (1.4%), ultrasound time (25 min), and extraction temperature (45°C). The highest yield of arctigenin, obtained under the optimal conditions was 6.39%, representing an increase of 28.15% compared to the reference extraction without enzyme processing. The IC 50 values of the extraction and arctigenin, respectively, for inhibiting AChE were 0.572 mg/ml and 0.462 mg/ml, and those for nitrite-scavenging were 34.571 mg/ml and 17.49 mg/ml. The results demonstrate that using an enzyme directly in the production is an effective means for extracting arctigenin from Fructus arctii. The extraction has the activities of inhibiting AChE and scavenging nitrite, probably because there has arctigenin in it. It is implied that the extraction and arctigenin could contribute to human health in clinical applications. The new method of adding enzyme directly to the preparation of arctigenin was carried out instead of preparing arctigenin by two

Novel Method of Preparation and Activity Research on Arctigenin from Fructus Arctii

PubMed Central

Cai, Enbo; Han, Jiahong; Yang, Limin; Zhang, Weiyuan; Zhao, Yan; Chen, Qiulian; Guo, Meng; He, Xinhong

2018-01-01

Background: Arctigenin has many pharmacological activities with clinical significance and is derived from Arctium lappa L. However, the present extraction method is inefficient and does not have meaningful industrial production. Objective: A new method to directly prepare arctigenin was established by combining enzyme-assisted extraction and central composite design. Arctigenin's further pharmacological activity was also surveyed in vitro. Materials and Methods: β-D-Glucosidase, a food-grade enzyme, was added directly to the fruits of A. lappa L. to hydrolyze the arctiin to arctigenin, and the obtained samples were subsequently subjected to ethanol (30%, v/v) extraction. The pharmacological activity of the extraction and arctigenin was determined by inhibiting acetylcholinesterase (AChE) and scavenging nitrite. Results: The factors investigated include the enzyme concentration (0.5%–2.5%), ultrasound time (10 min−3 0 min), and extraction temperature (30°C–50°C). From the analysis of the results by Design-Expert (V8.0.6), the optimal extraction conditions were obtained: enzyme concentration (1.4%), ultrasound time (25 min), and extraction temperature (45°C). The highest yield of arctigenin, obtained under the optimal conditions was 6.39%, representing an increase of 28.15% compared to the reference extraction without enzyme processing. The IC50 values of the extraction and arctigenin, respectively, for inhibiting AChE were 0.572 mg/ml and 0.462 mg/ml, and those for nitrite-scavenging were 34.571 mg/ml and 17.49 mg/ml. Conclusions: The results demonstrate that using an enzyme directly in the production is an effective means for extracting arctigenin from Fructus arctii. The extraction has the activities of inhibiting AChE and scavenging nitrite, probably because there has arctigenin in it. It is implied that the extraction and arctigenin could contribute to human health in clinical applications. SUMMARY The new method of adding enzyme directly to the

Impacts of oxidative stress on acetylcholinesterase transcription, and activity in embryos of zebrafish (Danio rerio) following Chlorpyrifos exposure.

PubMed

Rodríguez-Fuentes, Gabriela; Rubio-Escalante, Fernando J; Noreña-Barroso, Elsa; Escalante-Herrera, Karla S; Schlenk, Daniel

2015-01-01

Organophosphate pesticides cause irreversible inhibition of AChE which leads to neuronal overstimulation and death. Thus, dogma indicates that the target of OP pesticides is AChE, but many authors postulate that these compounds also disturb cellular redox processes, and change the activities of antioxidant enzymes. Interestingly, it has also been reported that oxidative stress plays also a role in the regulation and activity of AChE. The aims of this study were to determine the effects of the antioxidant, vitamin C (VC), the oxidant, t-butyl hydroperoxide (tBOOH) and the organophosphate Chlorpyrifos (CPF), on AChE gene transcription and activity in zebrafish embryos after 72h exposure. In addition, oxidative stress was evaluated by measuring antioxidant enzymes activities and transcription, and quantification of total glutathione. Apical effects on the development of zebrafish embryos were also measured. With the exception of AChE inhibition and enhanced gene expression, limited effects of CPF on oxidative stress and apical endpoints were found at this developmental stage. Addition of VC had little effect on oxidative stress or AChE, but increased pericardial area and heartbeat rate through an unknown mechanism. TBOOH diminished AChE gene expression and activity, and caused oxidative stress when administered alone. However, in combination with CPF, only reductions in AChE activity were observed with no significant changes in oxidative stress suggesting the adverse apical endpoints in the embryos may have been due to AChE inhibition by CPF rather than oxidative stress. These results give additional evidence to support the role of prooxidants in AChE activity and expression. Copyright © 2015 Elsevier Inc. All rights reserved.

Differential effects of lysophosphatidylcholine and ACh on muscarinic K(+),non-selective cation and Ca(2+) currents in guinea-pig atrial cells.

PubMed

Li, Libing; Matsuoka, Isao; Sakamoto, Kazuho; Kimura, Junko

2016-06-08

We compared the effects of lysophosphatidylcholine (LPC) and acetylcholine (ACh) on IK(ACh), ICa and a non-selective cation current (INSC) in guinea-pig atrial myocytes to clarify whether LPC and ACh activate similar Gi/o-coupled effector systems.　IK(ACh), ICa and INSC were analyzed in single atrial myocytes by the whole cell patch-clamp.　LPC induced INSC in a concentration-dependent manner in atrial cells.　ACh activated IK(ACh), but failed to evoke INSC.　LPC also activated IK(ACh) but with significantly less potency than ACh.　The effects of both ligands on IK(ACh) were inhibited by intracellular loading of pre-activated PTX.　This treatment also inhibited LPC-induced INSC, indicating that IK(ACh) and INSC induced by LPC are both mediated by Gi/o.　LPC and ACh had similar potencies in inhibiting ICa, which was pre-augmented by forskolin, indicating that LPC and ACh activate similar amounts of α-subunits of Gi/o.　The different effects of LPC and ACh on IK(ACh) and INSC may suggest that LPC and ACh activate Gi/o having different types of βγ subunits, and that LPC-induced INSC may be mediated by βγ subunits of Gi/o, which are less effective in inducing IK(ACh).

Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity.

PubMed

Miller, K W; Schamber, R; Osmanagaoglu, O; Ray, B

1998-06-01

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.

Response of digestive enzymes and esterases of ecotoxicological concern in earthworms exposed to chlorpyrifos-treated soils.

PubMed

Sanchez-Hernandez, Juan C; Ríos, Juan Manuel; Attademo, Andrés M

2018-03-01

Assessment of organophosphorus (OP) pesticide exposure in non-target organisms rarely involves non-neural molecular targets. Here we performed a 30-d microcosm experiment with Lumbricus terrestris to determine whether the activity of digestive enzymes (phosphatase, β-glucosidase, carboxylesterase and lipase) was sensitive to chlorpyrifos (5 mg kg -1 wet soil). Likewise, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the wall muscle and gastrointestinal tissues as indicators of OP exposure. Chlorpyrifos inhibited the acid phosphatase (34% of controls), carboxylesterase (25.6%) and lipase activities (31%) in the gastrointestinal content. However, in the gastrointestinal tissue, only the carboxylesterase and lipase activities were significantly depressed (42-67% carboxylesterase inhibition in the foregut and crop/gizzard, and 15% lipase inhibition in the foregut). Chlorpyrifos inhibited the activity of both cholinesterases in the gastrointestinal tissues, whereas the AChE activity was affected in the wall muscle. These results suggested chlorpyrifos was widely distributed throughout the earthworm body after 30 d of incubation. Interestingly, we found muscle carboxylesterase activity strongly inhibited (92% of control) compared with that detected in the gastrointestinal tissues of the same OP-exposed individuals. This finding was explained by the occurrence of pesticide-resistant esterases in the gastrointestinal tissues, which were evidenced by zymography. Our results suggest that digestive processes of L. terrestris may be altered by chlorpyrifos, as a consequence of the inhibitory action of the insecticide on some digestive enzymes.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Aiqiong; Du, Dan; Lin, Yuehe

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 detectionmore » 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.« less

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

Inhibition properties of propolis extracts to some clinically important enzymes.

PubMed

Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

2016-01-01

The present study was conducted to envisage inhibition effects of propolis on the crucial enzymes, urease, xanthine oxidase (XO) and acetylcholinesterase (AChE). Some of the antioxidant properties of the propolis samples were determined using the total phenolic content (TPE) and total flavonoids in the eight different ethanolic propolis extracts (EPE) samples. Inhibition values of the enzymes were expressed as inhibition concentration (IC 50 ; mg/mL or μg/mL) causing 50% inhibition of the enzymes with donepezil, acetohydroxamic acid and allopurinol as reference inhibitors. All the propolis extracts exhibited variable inhibition effects on these enzymes, but the higher the phenolic contents the lower the inhibitions values (IC 50 = 0.074 to 1.560 mg/mL). IC 50 values of the P5 propolis sample having the highest TPE, obtained from Zonguldak, for AChE, urease and XO were 0.081 ± 0.009, 0.080 ± 0.006 and 0.074 ± 0.011 μg/mL, respectively. The EPE proved to be a good source of inhibitor agents that can be used as natural inhibitors to serve human health.

Revealing the importance of linkers in K-series oxime reactivators for tabun-inhibited AChE using quantum chemical, docking and SMD studies.

PubMed

Ghosh, Shibaji; Chandar, Nellore Bhanu; Jana, Kalyanashis; Ganguly, Bishwajit

2017-08-01

Inhibition of acetylcholinesterase (AChE) with organophosphorus compounds has a detrimental effect on human life. Oxime K203 seems to be one of the promising reactivators for tabun-inhibited AChE than (K027, K127, and K628). These reactivators differ only in the linker units between the two pyridinium rings. The conformational analyses performed with quantum chemical RHF/6-31G* level for K027, K127, K203 and K628 showed that the minimum energy conformers have different orientations of the active and peripheral pyridinium rings for these reactivator molecules. K203 with (-CH 2 -CH=CH-CH 2 -) linker unit possesses more open conformation compared to the other reactivators. Such orientation of K203 experiences favorable interaction with the surrounding residues of catalytic anionic site (CAS) and peripheral anionic site (PAS) of tabun-inhibited AChE. From the steered molecular dynamics simulations, it has been observed that the oxygen atom of the oxime group of K203 reactivator approaches nearest to the P-atom of the SUN203 (3.75 Å) at lower time scales (less than ~1000 ps) as compared to the other reactivators. K203 experiences less number of hydrophobic interaction with the PAS residues which is suggested to be an important factor for the efficient reactivation process. In addition, K203 crates large number of H-bonding with CAS residues SUN203, Phe295, Tyr337, Phe338 and His447. K203 barely changes its conformation during the SMD simulation process and hence the energy penalty to adopt any other conformation is minimal in this case as compared to the other reactivators. The molecular mechanics and Poisson-Boltzmann surface area binding energies obtained for the interaction of K203 inside the gorge of tabun inhibited AChE is substantially higher (-290.2 kcal/mol) than the corresponding K628 reactivator (-260.4 kcal/mol), which also possess unsaturated aromatic linker unit.

Revealing the importance of linkers in K-series oxime reactivators for tabun-inhibited AChE using quantum chemical, docking and SMD studies

NASA Astrophysics Data System (ADS)

Ghosh, Shibaji; Chandar, Nellore Bhanu; Jana, Kalyanashis; Ganguly, Bishwajit

2017-08-01

Inhibition of acetylcholinesterase (AChE) with organophosphorus compounds has a detrimental effect on human life. Oxime K203 seems to be one of the promising reactivators for tabun-inhibited AChE than (K027, K127, and K628). These reactivators differ only in the linker units between the two pyridinium rings. The conformational analyses performed with quantum chemical RHF/6-31G* level for K027, K127, K203 and K628 showed that the minimum energy conformers have different orientations of the active and peripheral pyridinium rings for these reactivator molecules. K203 with (-CH2-CH=CH-CH2-) linker unit possesses more open conformation compared to the other reactivators. Such orientation of K203 experiences favorable interaction with the surrounding residues of catalytic anionic site (CAS) and peripheral anionic site (PAS) of tabun-inhibited AChE. From the steered molecular dynamics simulations, it has been observed that the oxygen atom of the oxime group of K203 reactivator approaches nearest to the P-atom of the SUN203 (3.75 Å) at lower time scales (less than 1000 ps) as compared to the other reactivators. K203 experiences less number of hydrophobic interaction with the PAS residues which is suggested to be an important factor for the efficient reactivation process. In addition, K203 crates large number of H-bonding with CAS residues SUN203, Phe295, Tyr337, Phe338 and His447. K203 barely changes its conformation during the SMD simulation process and hence the energy penalty to adopt any other conformation is minimal in this case as compared to the other reactivators. The molecular mechanics and Poisson-Boltzmann surface area binding energies obtained for the interaction of K203 inside the gorge of tabun inhibited AChE is substantially higher (-290.2 kcal/mol) than the corresponding K628 reactivator (-260.4 kcal/mol), which also possess unsaturated aromatic linker unit.

Antioxidant and acetylcholinesterase inhibitory activities of ginger root (Zingiber officinale Roscoe) extract.

PubMed

Tung, Bui Thanh; Thu, Dang Kim; Thu, Nguyen Thi Kim; Hai, Nguyen Thanh

2017-05-04

Background Zingiber officinale Roscoe has been used in traditional medicine for the treatment of neurological disorder. This study aimed to investigate the phenolic contents, antioxidant, acetylcholinesterase enzyme (AChE) inhibitory activities of different fraction of Z. officinale root grown in Vietnam. Methods The roots of Z. officinale are extracted with ethanol 96 % and fractionated with n-hexane, ethyl acetate (EtOAc) and butanol (BuOH) solvents. These fractions evaluated the antioxidant activity by 1,1-Diphenyl -2-picrylhydrazyl (DPPH) assay and AChE inhibitory activity by Ellman's colorimetric method. Results Our data showed that the total phenolic content of EtOAc fraction was highest equivalents to 35.2±1.4 mg quercetin/g of fraction. Our data also demonstrated that EtOAc fraction had the strongest antioxidant activity with IC50 was 8.89±1.37 µg/mL and AChE inhibitory activity with an IC50 value of 22.85±2.37 μg/mL in a dose-dependent manner, followed by BuOH fraction and the n-hexane fraction is the weakest. Detailed kinetic analysis indicated that EtOAc fraction was mixed inhibition type with Ki (representing the affinity of the enzyme and inhibitor) was 30.61±1.43 µg/mL. Conclusions Our results suggest that the EtOAc fraction of Z. officinale may be a promising source of AChE inhibitors for Alzheimer's disease.

Phenolic Extracts from Clerodendrum volubile Leaves Inhibit Cholinergic and Monoaminergic Enzymes Relevant to the Management of Some Neurodegenerative Diseases.

PubMed

Oboh, Ganiyu; Ogunruku, Omodesola O; Oyeleye, Sunday I; Olasehinde, Tosin A; Ademosun, Ayokunle O; Boligon, Aline Augusti

2017-05-04

This study investigated the inhibitory effects of phenolic-rich extracts from Clerodendrum volubile leaves on cholinergic [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] and monoaminergic [monoamine oxidase (MAO)] enzymes' activities and pro-oxidants [Fe 2+ and quinolinic acid-(QA)] induced lipid peroxidation in rats brain homogenates in vitro. Free phenolic extracts (FPE) and bound phenolic extracts (BPE) were obtained via solvent extraction, and the total phenol and flavonoid contents were evaluated. The phenolic constituents of the extracts were also determined using high performance liquid chromatography coupled with diode array detector (HPLC-DAD). Our findings revealed that FPE had higher AChE (2.06 μg/mL), BChE (2.79 μg/mL), and MAO (2.81 μg/mL) inhibitory effects than BPE [AChE, 2.80 μg/mL; BChE, 3.40 μg/mL; MAO, 3.39 μg/mL]. Furthermore, FPE also had significantly (P < 0.05) higher inhibitory effects on Fe 2+ and QA-induced lipid peroxidation compared to BPE. FPE (162.61 mg GAE/g) had higher total phenol content than BPE. However, BPE (18.65 mg QE/g) had significantly higher total flavonoid content than FPE (13.32 mg QE/g). Phenolic acids (such as gallic acid, catechin, chlorogenic, caffeic, ellagic, p-Coumaric acids) and flavonoids (catechins, rutin and quercetin) were present in both extracts. This study revealed that the enzymes' inhibitory activities and antioxidant potentials of phenolic-rich extracts from C. volubile could be part of the mechanism of actions behind its use for memory/cognitive function as obtained in folklore. However, FPE exhibited significantly higher enzymes, inhibitory and antioxidant potentials than BPE.

Screening for antimalarial and acetylcholinesterase inhibitory activities of some Iranian seaweeds

PubMed Central

Ghannadi, A; Plubrukarn, A; Zandi, K; Sartavi, K; Yegdaneh, A

2013-01-01

Alcoholic extracts of 8 different types of seaweeds from Iran’s Persian Gulf were tested for their antimalarial and acetylcholinesterase enzyme (AChE) inhibitory activities for the first time. A modified Ellman and Ingkaninan method was used for measuring AChE inhibitory activity in which galanthamine was used as the reference. The antimalarial assay was performed using microculture radioisotope technique. Mefloquine and dihydroartemisinin were uased as the standards. The extract of Sargassum boveanum (Sargasseae family) showed the highest AChE inhibitory activity (IC50 equals to 1 mg ml-1) while Cystoseira indica (Cystoseiraceae family) exhibited the least activity (IC50 of 11 mg ml-1). The species from Rhodophyta (Gracilaria corticata and Gracilaria salicornia) also showed moderate activities (IC509.5, 8.7 mg ml-1, respectively). All extracts were inactive in antimalarial assay. PMID:24019820

A multi-enzyme microreactor-based online electrochemical system for selective and continuous monitoring of acetylcholine.

PubMed

Lin, Yuqing; Yu, Ping; Mao, Lanqun

2015-06-07

This study demonstrates an online electrochemical system (OECS) for selective and continuous measurements of acetylcholine (ACh) through efficiently integrating in vivo microdialysis, a multi-enzyme microreactor and an electrochemical detector. A multi-enzyme microreactor was prepared first by co-immobilizing two kinds of enzymes, i.e. choline oxidase (ChOx) and catalase (Cat), onto magnetite nanoparticles and then confining the as-formed nanoparticles into a fused-silica capillary with the assistance of an external magnet. The multi-enzyme microreactor was settled between an in vivo microdialysis sampling system and an electrochemical detector to suppress the interference from choline toward ACh detection. Selective detection of ACh was accomplished using the electrochemical detector with ACh esterase (AChE) and ChOx as the recognition units for ACh and Prussian blue (PB) as the electrocatalyst for the reduction of hydrogen peroxide (H2O2). The current recorded with the OECS was linear with the concentration of ACh (I/nA = -3.90CACh/μM + 1.21, γ = 0.998) within a concentration range of 5 μM to 100 μM. The detection limit, based on a signal-to-noise ratio of 3, was calculated to be 1 μM. Interference investigation demonstrates that the OECS did not produce an observable current response toward physiological levels of common electroactive species, such as ascorbic acid (AA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and uric acid (UA). The high selectivity and the good linearity in combination with the high stability may enable the OECS developed here as a potential system for continuous monitoring of cerebral ACh release in some physiological and pathological processes.

Neurotoxic responses in brain tissues of rainbow trout exposed to imidacloprid pesticide: Assessment of 8-hydroxy-2-deoxyguanosine activity, oxidative stress and acetylcholinesterase activity.

PubMed

Topal, Ahmet; Alak, Gonca; Ozkaraca, Mustafa; Yeltekin, Aslı Cilingir; Comaklı, Selim; Acıl, Gurdal; Kokturk, Mine; Atamanalp, Muhammed

2017-05-01

The extensive use of imidacloprid, a neonicotinoid insecticide, causes undesirable toxicity in non-targeted organisms including fish in aquatic environments. We investigated neurotoxic responses by observing 8-hydroxy-2-deoxyguanosine (8-OHdG) activity, oxidative stress and acetylcholinesterase (AChE) activity in rainbow trout brain tissue after 21 days of imidacloprid exposure at levels of (5 mg/L, 10 mg/L, 20 mg/L). The obtained results indicated that 8-OHdG activity did not change in fish exposed to 5 mg/L of imidacloprid, but 10 mg/L and 20 mg/L of imidacloprid significantly increased 8-OHdG activity compared to the control (p < 0.05). An immunopositiv reaction to 8-OHdG was detected in brain tissues. The brain tissues indicated a significant increase in antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) compared to the control and there was a significant increase in malondialdehyde (MDA) levels (p < 0.05). High concentrations of imidacloprid caused a significant decrease in AChE enzyme activity (p < 0.05). These results suggested that imidacloprid can be neurotoxic to fish by promoting AChE inhibition, an increase in 8-OHdG activity and changes in oxidative stress parameters. Therefore, these data may reflect one of the molecular pathways that play a role in imidacloprid toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in antioxidant status, protein concentration, acetylcholinesterase, (Na+,K+)-, and Mg2+ -ATPase activities in the brain of hyper- and hypothyroid adult rats.

PubMed

Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Mourouzis, Iordanis; Varonos, Dennis; Cokkinos, Dennis; Tsakiris, Stylianos

2005-06-01

It is a common knowledge that metabolic reactions increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how the metabolic reactions could affect the total antioxidant status (TAS), protein concentration (PC) and the activities of acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+ -ATPase in the brain of hyper- and hypothyroid adult male rats. Hyperthyroidism was induced in rats by subcutaneous administration of thyroxine (25 microg/l00 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. TAS, PC, and enzyme activities were evaluated spectrophotometrically in the homogenated brain of each animal. TAS, PC, and Mg2+ -ATPase activity were found unaffected in hyperthyroidism, while AChE and Na+,K+ -ATPase activities were reduced by 25% (p < 0.01). In contrast, TAS, (Na+,K+)-ATPase and Mg2+-ATPase activities were found to be increased (approx. 23-30%, p < 0.001) in the hypothyroid brain, while AChE activity and PC were shown to be inhibited (approx. 23-30%, p < 0.001). These changes on brain enzyme activities may reflect the different metabolic effects of hyper- and hypothyroidism. Such changes of the enzyme activities may differentially modulate the brain intracellular Mg2+, neural excitability, as well as the uptake and release of biogenic amines.

α7nAchR/NMDAR coupling affects NMDAR function and object recognition.

PubMed

Li, Shupeng; Nai, Qiang; Lipina, Tatiana V; Roder, John C; Liu, Fang

2013-12-20

The α7 nicotinic acetylcholine receptor (nAchR) and NMDA glutamate receptor (NMDAR) are both ligand-gated ion channels permeable to Ca2+ and Na+. Previous studies have demonstrated functional modulation of NMDARs by nAchRs, although the molecular mechanism remains largely unknown. We have previously reported that α7nAchR forms a protein complex with the NMDAR through a protein-protein interaction. We also developed an interfering peptide that is able to disrupt the α7nAchR-NMDAR complex and blocks cue-induced reinstatement of nicotine-seeking in rat models of relapse. In the present study, we investigated whether the α7nAchR-NMDAR interaction is responsible for the functional modulation of NMDAR by α7nAchR using both electrophysiological and behavioral tests. We have found that activation of α7nAchR upregulates NMDAR-mediated whole cell currents and LTP of mEPSC in cultured hippocampal neurons, which can be abolished by the interfering peptide that disrupts the α7nAchR-NMDAR interaction. Moreover, administration of the interfering peptide in mice impairs novel object recognition but not Morris water maze performance. Our results suggest that α7nAchR/NMDAR coupling may selectively affect some aspects of learning and memory.

Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

PubMed

Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

2016-01-01

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

Muscarinic ACh Receptors Contribute to Aversive Olfactory Learning in Drosophila.

PubMed

Silva, Bryon; Molina-Fernández, Claudia; Ugalde, María Beatriz; Tognarelli, Eduardo I; Angel, Cristian; Campusano, Jorge M

2015-01-01

The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, we have advanced our understanding on the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. Here we evaluate for the first time the proposition that, as in mammals, muscarinic ACh receptors (mAChRs) contribute to memory formation in Drosophila. Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila.

Muscarinic ACh Receptors Contribute to Aversive Olfactory Learning in Drosophila

PubMed Central

Silva, Bryon; Molina-Fernández, Claudia; Ugalde, María Beatriz; Tognarelli, Eduardo I.; Angel, Cristian; Campusano, Jorge M.

2015-01-01

The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, we have advanced our understanding on the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. Here we evaluate for the first time the proposition that, as in mammals, muscarinic ACh receptors (mAChRs) contribute to memory formation in Drosophila. Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila. PMID:26380118

Acetylcholine ameliorates endoplasmic reticulum stress in endothelial cells after hypoxia/reoxygenation via M3 AChR-AMPK signaling.

PubMed

Bi, Xueyuan; He, Xi; Xu, Man; Zhao, Ming; Yu, Xiaojiang; Lu, Xingzhu; Zang, Weijin

2015-08-03

Endoplasmic reticulum (ER) stress is associated with various cardiovascular diseases. However, its pathophysiological relevance and the underlying mechanisms in the context of hypoxia/reoxygenation (H/R) in endothelial cells are not fully understood. Previous findings have suggested that acetylcholine (ACh), the major vagal nerve neurotransmitter, protected against cardiomyocyte injury by activating AMP-activated protein kinase (AMPK). This study investigated the role of ER stress in endothelial cells during H/R and explored the beneficial effects of ACh. Our results showed that H/R triggered ER stress and apoptosis in endothelial cells, evidenced by the elevation of glucose-regulated protein 78, cleaved caspase-12 and C/EBP homologous protein expression. ACh significantly decreased ER stress and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling positive cells and restored ER ultrastructural changes induced by H/R, possibly via protein kinase-like ER kinase and inositol-requiring kinase 1 pathways. Additionally, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3 AChR) inhibitor, abolished ACh-mediated increase in AMPK phosphorylation during H/R. Furthermore, M3 AChR or AMPK siRNA abrogated the ACh-elicited the attenuation of ER stress in endothelial cells, indicating that the salutary effects of ACh were likely mediated by M3 AChR-AMPK signaling. Overall, ACh activated AMPK through M3 AChR, thereby inhibited H/R-induced ER stress and apoptosis in endothelial cells. We have suggested for the first time that AMPK may function as an essential intermediate step between M3 AChR stimulation and inhibition of ER stress-associated apoptotic pathway during H/R, which may help to develop novel therapeutic approaches targeting ER stress to prevent or alleviate ischemia/reperfusion injury.

Biophenols: Enzymes (β-secretase, Cholinesterases, histone deacetylase and tyrosinase) inhibitors from olive (Olea europaea L.).

PubMed

Omar, Syed Haris; Scott, Christopher J; Hamlin, Adam S; Obied, Hassan K

2018-07-01

The focus of this study was on inhibition of enzymes involved in the pathogenesis Alzheimer's disease (AD) including prime amyloid beta (Aβ) producing enzyme (β-secretase: BACE-1) and disease progression enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), histone deacetylase (HDAC), and tyrosinase along with the catecholamine L-DOPA, by using olive biophenols. Here we report the strongest inhibition of BACE-1 from rutin (IC 50 : 3.8 nM) followed by verbascoside (IC 50 : 6.3 nM) and olive fruit extract (IC 50 : 18 ng), respectively. Olive biophenol, quercetin exhibited strongest enzyme inhibitory activity against tyrosinase (IC 50 : 10.73 μM), BChE (IC 50 : 19.08 μM), AChE (IC 50 : 55.44 μM), and HDAC (IC 50 : 105.1 μM) enzymes. Furthermore, olive biophenol verbascoside (IC 50 : 188.6 μM), and hydroxytyrosol extreme extract (IC 50 : 66.22 μg) were showed the highest levels of inhibition against the HDAC enzyme. Neuroprotective capacity against levodopa-induced toxicity in neuroblastoma (SH-SY5Y) cells of olive biophenols were assessed, where rutin indicated the highest neuroprotection (74%), followed by caffeic acid (73%), and extract hydroxytyrosol extreme (97%), respectively. To the best of our knowledge, this is the first in vitro report on the enzymes inhibitory activity of olive biophenols. Taken together, our in vitro results data suggest that olive biophenols could be a promising natural inhibitor, which may reduce the enzyme-induced toxicity associated with the oxidative stress involved in the progression of AD. Acetylthiocholine iodide (PubChem CID: 74629); S-Butyrylthiocholine chloride (PubChem CID: 3015121); Caffeic acid (PubChem CID: 689043); Dimethyl sulfoxide (DMSO) (PubChem: 679); L-3,4-Dihydroxyphenylalanine (L-DOPA) (PubChem CID: 6047); 5,5'-Dithiobis (2-nitrobenzoic acid) (DTNB) (PubChem CID: 6254); Epigallocatechin gallate (EGCG) (PubChem CID: 65064); Ethylenediamine tetraacetic acid (EDTA) (Pub

Differential effects of developmental hypo- and hyperthyroidism on acetylcholinesterase and butyrylcholinesterase activity in the spinal cord of developing postnatal rat pups.

PubMed

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

2012-11-01

The plasticity and vulnerability of the rat spinal cord (SC) during postnatal development has been less investigated compared to other CNS structures. In this study, we determined the effects of thyroid hormonal (TH) deficiency and excess on postnatal growth and neurochemical development of the rat SC. The growth as well as the specific and total activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes of the SC were determined in hypo- and hyperthyroid rat pups at postnatal (P) days P1, P5, P10 and P21 (weaning), and were compared to age-matched untreated normal controls. AChE is a cholinergic synaptic enzyme while BuChE is a metabolic enzyme mainly found in glial cells and neurovascular cells. The SC is rich in somatic motor, autonomic cholinergic neurons and associated interneurons. Daily subcutaneous injection of pups with thyroxine (T4) and administration of antithyroid goitrogen propylthiouracil (PTU) in the litter's drinking water were used to induce hyper- and hypothyroidism, respectively. Enzyme assays were carried out spectrophotometrically at the above-mentioned ages, using SC homogenates with acetylthiocholine-chloride as the substrate, together with specific cholinesterase inhibitors, which specifically target AChE and BuChE. SC weights were significantly lower at P10 and P21 in hypothyroid pups but unchanged in the hyperthyroid ones. Hypothyroidism significantly reduced both specific and total AChE activity in SC of P10 and P21 rat pups, while having no effects on the BuChE activity, although total BuChE activity was decreased due to reduced total tissue weight. In contrast both specific and total AChE activities were markedly and significantly increased (>100%) in the P10 and P21 hyperthyroid pups. However, BuChE specific activity was unaffected by this treatment. The results indicate that hypothyroid condition significantly reduces, while hyperthyroidism increases, the postnatal development of cholinergic synapses, thereby

Alkaloids from roots of Stephania rotunda and their cholinesterase inhibitory activity.

PubMed

Hung, Tran Manh; Dang, Nguyen Hai; Kim, Jin Cheol; Jang, Han-Su; Ryoo, Sung-Woo; Lee, Jeong Hyung; Choi, Jae Sue; Bae, Kihwan; Min, Byung Sun

2010-10-01

In the course of screening plants used in folk medicine as memory enhancers, a 70% ethanolic extract of Stephania rotunda roots showed significant AChE inhibitory activity. Repeated column chromatography led to the isolation of a new protoberberine alkaloid, which we named stepharotudine (1), and seven known compounds (2-8). The chemical structures of the isolated compounds were elucidated based on extensive 1D and 2D NMR spectroscopic data. Compounds 1-8 were investigated in vitro for their anticholinesterase activity using a rat cortex AChE enzyme. © Georg Thieme Verlag KG Stuttgart · New York.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

NASA Astrophysics Data System (ADS)

Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Gurung, Arun Bahadur; Bhattacharjee, Atanu; Aguan, Kripamoy; Mitra, Sivaprasad

2018-01-01

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction

PubMed Central

Ghazanfari, Nazanin; Morsch, Marco; Reddel, Stephen W; Liang, Simon X; Phillips, William D

2014-01-01

Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold. PMID:24860174

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.

PubMed

Ghazanfari, Nazanin; Morsch, Marco; Reddel, Stephen W; Liang, Simon X; Phillips, William D

2014-07-01

Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Inkjet-assisted layer-by-layer printing of quantum dot/enzyme microarrays for highly sensitive detection of organophosphorous pesticides.

PubMed

Luan, Enxiao; Zheng, Zhaozhu; Li, Xinyu; Gu, Hongxi; Liu, Shaoqin

2016-04-15

We present a facile fabrication of layer-by-layer (LbL) microarrays of quantum dots (QDs) and acetylcholinesterase enzyme (AChE). The resulting arrays had several unique properties, such as low cost, high integration and excellent flexibility and time-saving. The presence of organophosphorous pesticides (OPs) can inhibit the AChE activity and thus changes the fluorescent intensity of QDs/AChE microscopic dot arrays. Therefore, the QDs/AChE microscopic dot arrays were used for the sensitive visual detection of OPs. Linear calibration for parathion and paraoxon was obtained in the range of 5-100 μg L(-1) under the optimized conditions with the limit of detection (LOD) of 10 μg L(-1). The arrays have been successfully used for detection of OPs in fruits and water real samples. The new array was validated by comparison with conventional high performance liquid chromatography-mass spectrometry (HPLC-MS). Copyright © 2016 Elsevier B.V. All rights reserved.

Design, synthesis and preliminary structure-activity relationship investigation of nitrogen-containing chalcone derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors: a further study based on Flavokawain B Mannich base derivatives.

PubMed

Liu, Haoran; Fan, Haoqun; Gao, Xiaohui; Huang, Xueqing; Liu, Xianjun; Liu, Linbo; Zhou, Chao; Tang, Jingjing; Wang, Qiuan; Liu, Wukun

2016-08-01

In order to study the structure-activity relationship of Flavokawain B Mannich-based derivatives as acetylcholinesterase (AChE) inhibitors in our recent investigation, 20 new nitrogen-containing chalcone derivatives (4 a-8d) were designed, synthesized, and evaluated for AChE inhibitory activity in vitro. The results suggested that amino alkyl side chain of chalcone dramatically influenced the inhibitory activity against AChE. Among them, compound 6c revealed the strongest AChE inhibitory activity (IC50 value: 0.85 μmol/L) and the highest selectivity against AChE over BuChE (ratio: 35.79). Enzyme kinetic study showed that the inhibition mechanism of compound 6c against AChE was a mixed-type inhibition. The molecular docking assay showed that this compound can both bind with the catalytic site and the peripheral site of AChE.

High aryl acylamidase activity associated with cobra venom acetylcholinesterase: biological significance.

PubMed

Rajesh, Ramanna V; Layer, Paul G; Boopathy, Rathanam

2009-01-01

Investigation of the non-classical functions of cholinesterases (ChEs) has been the subject of interest in the past three decades. One of which is aryl acylamidase (AAA) activity associated with ChEs, but characterized in in vitro, as an enzyme, splitting the artificial substrate o-nitroacetanilide with unknown physiological function. In the present study, we have compared levels of AAA activity of AChE from different sources like goat brain, electric eel organ and from venoms of different snakes. Remarkably cobra venom showed the highest AAA activity and also high AAA/AChE ratio. Both serotonergenic and cholinergic inhibitors inhibited the cobra venom AAA activity in a concentration dependent manner, which also underlines the association of AAA with AChE of cobra venom. The study becomes interesting because of i) the cobra venom AChE exists in monomeric globular forms; ii) in Alzheimer's disease too the most abundant forms of cholinesterases are monomeric globular forms, thought to be involved in the pathogenesis of Alzheimer's disease; iii) the effect of Alzheimer's disease drugs on the AAA activity of cobra venom, indicated that AAA activity of cobra venom was more sensitive than AChE and iv) Huperzine and Tacrine showed more pronounced effect on AAA. Thus, this study elucidates the high AAA associated with cobra venom AChE may serve as one of the prominent activity to test the pharmacological effect of AD drugs, as other sources were found to have lower activity.

Voltage sensitivity of M2 muscarinic receptors underlies the delayed rectifier-like activation of ACh-gated K(+) current by choline in feline atrial myocytes.

PubMed

Navarro-Polanco, Ricardo A; Aréchiga-Figueroa, Iván A; Salazar-Fajardo, Pedro D; Benavides-Haro, Dora E; Rodríguez-Elías, Julio C; Sachse, Frank B; Tristani-Firouzi, Martin; Sánchez-Chapula, José A; Moreno-Galindo, Eloy G

2013-09-01

Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). In canine and guinea pig atrial myocytes, Ch was shown to activate an outward K(+) current in a delayed rectifier fashion. This current has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the exact nature and identity of this current has not been convincingly established. We recently described the unique ligand- and voltage-dependent properties of muscarinic activation of ACh-activated K(+) current (IKACh) and showed that, in contrast to ACh, pilocarpine induces a current with delayed rectifier-like properties with membrane depolarization. Here, we tested the hypothesis that Ch activates IKACh in feline atrial myocytes in a voltage-dependent manner similar to pilocarpine. Single-channel recordings, biophysical profiles, specific pharmacological inhibition and computational data indicate that the current activated by Ch is IKACh. Moreover, we show that membrane depolarization increases the potency and efficacy of IKACh activation by Ch and thus gives the appearance of a delayed rectifier activating K(+) current at depolarized potentials. Our findings support the emerging concept that IKACh modulation is both voltage- and ligand-specific and reinforce the importance of these properties in understanding cardiac physiology.

Cognitive enhancing effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers on learning and memory

PubMed Central

Nade, V. S.; Kawale, L. A.; Valte, K. D.; Shendye, N. V.

2015-01-01

Objective: The present study was designed to investigate cognitive enhancing property of angiotensin-converting enzymes inhibitors (ACEI) and angiotensin receptor blockers (ARBs) in rats. Materials and Methods: The elevated plus maze (EPM), passive avoidance test (PAT), and water maze test (WMT) were used to assess cognitive enhancing activity in young and aged rats. Ramipril (10 mg/kg, p.o.), perindopril (10 mg/kg, i.p), losartan (20 mg/kg, i.p), and valsartan (20 mg/kg, p.o) were administered to assess their effect on learning and memory. Scopolamine (1 mg/kg, i.p) was used to impair cognitive function. Piracetam (200 mg/kg, i.p) was used as reference drug. Results: All the treatments significantly attenuated amnesia induced by aging and scopolamine. In EPM, aged and scopolamine-treated rats showed an increase in transfer latency (TL) whereas, ACEI and ARBs showed a significant decrease in TL. Treatment with ACEI and ARBs significantly increased step down latencies and decreased latency to reach the platform in target quadrant in young, aged and scopolamine-treated animals in PAT and WMT, respectively. The treatments inhibited acetylcholinesterase (AChE) enzyme in the brain. Similarly, all the treatments attenuated scopolamine-induced lipid peroxidation and normalize antioxidant enzymes. Conclusion: The results suggest that the cognitive enhancing effect of ACEI and ARBs may be due to inhibition of AChE or by regulation of antioxidant system or increase in formation of angiotensin IV. PMID:26069362

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

PubMed

Bate, Paul; Warwicker, Jim

2004-07-02

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

Alteration of paraoxonase, arylesterase and lactonase activities in people around fluoride endemic area of Tamil Nadu, India.

PubMed

Arulkumar, Mani; Vijayan, Raji; Penislusshiyan, Sakayanathan; Sathishkumar, Palanivel; Angayarkanni, Jayaraman; Palvannan, Thayumanavan

2017-08-01

Toxicity due to excess fluoride concentration in drinking water is of great concern in people who rely only on the ground water as their water source in many region of the world. We collected samples and examined the toxicity of fluoride in a population residing at Salem, Dharmapuri and Krishnagiri districts of Tamil Nadu, India and measured HDL bound enzyme (PON1), erythrocyte membrane bound enzymes (acetylcholinesterase, AChE) and adenosine 5' triphosphatase (ATPases), plasma enzyme (butyrylcholinesterase, BChE) and rate limiting enzyme in heme biosynthesis (delta aminolevulinic acid dehydratase, δ-ALAD) activities. In fluorosis patients, formation of lipid peroxidation product was more in erythrocytes than in plasma. The observation further revealed that there was 50% reduction in the activity of HDL bound anti atherogenic enzyme-paraoxonase (PON1). The activities of membrane bound and signaling enzymes (acetylcholinesterase - AChE and adenosine 5' triphosphatase - ATPase) of erythrocyte were also diminished. These results suggested that there was defectiveness in the signaling and energy metabolism in fluorosis patients. Altered isoenzyme pattern of lactate dehydrogenase (LDH) in fluorosis samples was observed. Furthermore, the result suggested that both the heart (LDH 1) and liver (LDH 5) were most affected by fluoride toxicity. The study also provided reference values for tests which are used to predict the severity of fluoride toxicity. The toxic effect of fluoride was due to the collective effects on vital protective system rather than single factor. Copyright © 2017 Elsevier B.V. All rights reserved.

An ex vivo study of nitric oxide efflux from human erythrocytes in both genders.

PubMed

Duarte, Catarina; Napoleão, Patrícia; Freitas, Teresa; Saldanha, Carlota

2016-01-01

Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux. This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females. We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity. There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p < 0.05) in women than in men. The efflux of NO from erythrocyte of healthy humans did not change with gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

Myasthenia Gravis and the Tops and Bottoms of AChRs Antigenic Structure of the MIR and Specific Immunosuppression of EAMG Using AChR Cytoplasmic Domains

PubMed Central

Lindstrom, Jon; Luo, Jie; Kuryatov, Alexander

2009-01-01

The main immunogenic region (MIR), against which half or more of the autoantibodies to acetylcholine receptors (AChRs) in myasthenia gravis (MG) or experimental autoimmune MG (EAMG) are directed, is located at the extracellular end of α1 subunits. Rat monoclonal antibodies (mAbs) to the MIR efficiently compete with MG patient autoantibodies for binding to human muscle AChRs. Antibodies bound to the MIR do not interfere with cholinergic ligand binding or AChR function, but target complement and trigger antigenic modulation. Rat mAbs to the MIR also bind to human ganglionic AChR α3 subunits, but MG patient antibodies do not. By making chimeras of α1 subunits with α7 subunits or ACh binding protein, the structure of the MIR and its functional effects are being investigated. Many mAbs to the MIR bind only to the native conformation of α1 subunits because they bind to sequences that are adjacent only in the native structure. The MIR epitopes recognized by these mAbs are not recognized by most patient antibodies whose epitopes must be nearby. The presence of the MIR epitopes in α1/α7 chimeras greatly promotes AChR expression and sensitivity to activation. EAMG can be suppressed by treatment with denatured, bacterially expressed mixtures of extracellular and cytoplasmic domains of human α1, β1, γ, δ, and ε subunits. A mixture of only the cytoplasmic domains not only avoids the potential liability of provoking formation antibodies to pathologically significant epitopes on the extracellular surface, but also potently suppresses the development of EAMG. PMID:18567851

Modulators of Acetylcholinesterase Activity: From Alzheimer's Disease to Anti-Cancer Drugs.

PubMed

Lazarevic-Pasti, Tamara; Leskovac, Andreja; Momic, Tatjana; Petrovic, Sandra; Vasic, Vesna

2017-01-01

Acetylcholinesterase (AChE) 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 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 for different neurodegenerative diseases (such as Alzheimer's and Parkinson's) as well as toxins. At the same time, there are increasing evidence that in non-neuronal context, AChE is involved in the regulation of cell proliferation, differentiation, apoptosis and cell-cell interaction. An irregular expression of AChE has been found in different types of tumors, suggesting the involvement of AChE in the regulation of tumor development. Having all this in mind, there is a possibility that some AChE inhibitors could be used as anti-cancer agents. This contribution will discuss a broad range of possible application of different AChE inhibitors as drugs, from well-known anti-Alzheimer's disease drugs to their use in cancer treatment in future. Emphasis will be put on various known AChE inhibitors classes, whose application as drugs could be controversy, as well as on newly investigated natural products, which can also modulate AChE activity. It is not clear a patient treated for neurodegenerative condition prone to increased risk for some types of cancer and vice versa. This is necessary to keep in mind during rational drug design process for all therapies, which are based on AChE as a target molecule. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Comparison of the oxime-induced reactivation of rhesus monkey, swine and guinea pig erythrocyte acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity.

PubMed

Herkert, Nadja M; Lallement, Guy; Clarençon, Didier; Thiermann, Horst; Worek, Franz

2009-04-28

Recently, a dynamically working in vitro model with real-time determination of membrane-bound human acetylcholinesterase (AChE) activity was shown to be a versatile model to investigate oxime-induced reactivation kinetics of organophosphate- (OP) inhibited enzyme. In this assay, AChE was immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. Subsequently, AChE activity was continuously analyzed in a flow-through detector. Now, it was an intriguing question whether this model could be used with erythrocyte AChE from other species in order to investigate kinetic interactions in the absence of annoying side reactions. Rhesus monkey, swine and guinea pig erythrocytes were a stable and highly reproducible enzyme source. Then, the model was applied to the reactivation of sarin- and paraoxon-inhibited AChE by obidoxime or HI 6 and it could be shown that the derived reactivation rate constants were in good agreement to previous results obtained from experiments with a static model. Hence, this dynamic model offers the possibility to investigate highly reproducible interactions between AChE, OP and oximes with human and animal AChE.

Plasma B-esterase activities in European raptors.

PubMed

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

2005-01-01

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

Paraoxonase (PON1) polymorphism and activity as the determinants of sensitivity to organophosphates in human subjects.

PubMed

Sirivarasai, Jintana; Kaojarern, Sming; Yoovathaworn, Krongtong; Sura, Thanyachai

2007-07-20

Paraoxonase (PON1) plays an important role in mechanism of organophosphorus compound (OP) toxicity, as seen both in vitro and in vivo studies. Polymorphisms of PON1 gene at coding and promoter regions have also been to affect on the hydrolytic activity and PON1 level. The objectives of this study were to determine PON1 polymorphism and activity in an OP-exposed population and the effects on inhibition of cholinesterase activity. The studied population consisted of control (n=30) and exposed groups (n=90). All enzyme activities (AChE, BuChE, paraoxonase, arylesterase and diazonase) were measured once for control group and two periods of exposure for exposed group. Three polymorphisms of PON1 (Q192R, L55M and T-108C) were identified only in the exposed subjects. The results demonstrated that AChE activity in both high (345.5 microkat/gHb) and low exposure periods (496.9 microkat/gHb) of the exposed group were significantly different from control group (649.7 microkat/gHb, p<0.01). For BuChE activity, the exposed group also showed the statistically lower level in both periods (high exposure period: 62.17 microkat/L and low exposure period: 81.84 microkat/L) than those in the control group (93.35 microkat/L). Serum paraoxonase activity was significantly different among individual genotypes, RR>QR>RR, LL>LM and -108CC>-108CT>-108TT, but this was not found for those of arylesterase and diazonase activities. Q192R and L55M as well as Q192R and T-108C also presented substantial linkage disequilibrium. Further analysis was performed with haplotypes and various enzyme activities. AChE activity was not affected by haplotypes. Individuals with "211" haplotype showed significantly higher paraoxonase activity and BuChE activity than other haplotypes but not in diazonase activity. In conclusion, PON1 gene exhibited a wide variation in enzyme activities both within and between genotypes which implied insights of a potentially difference in sensitivity to OP toxicity.

Preparation, anticholinesterase activity and molecular docking of new lupane derivatives.

PubMed

Castro, María Julia; Richmond, Victoria; Romero, Carmen; Maier, Marta S; Estévez-Braun, Ana; Ravelo, Angel G; Faraoni, María Belén; Murray, Ana Paula

2014-07-01

A set of twenty one lupane derivatives (2-22) was prepared from the natural triterpenoid calenduladiol (1) by transformations on the hydroxyl groups at C-3 and C-16, and also on the isopropenyl moiety. The derivatives were tested for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and some structure-activity relationships were outlined with the aid of enzyme kinetic studies and docking modelization. The most active compound resulted to be 3,16,30-trioxolup-20(29)-ene (22), with an IC50 value of 21.5μM for butyrylcholinesterase, which revealed a selective inhibitor profile towards this enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of caffeine, caffeic acid and their various combinations on enzymes of cholinergic, monoaminergic and purinergic systems critical to neurodegeneration in rat brain-In vitro.

PubMed

Akomolafe, S F; Akinyemi, A J; Ogunsuyi, O B; Oyeleye, S I; Oboh, G; Adeoyo, O O; Allismith, Y R

2017-09-01

Caffeine and caffeic acid are two bioactive compounds that are present in plant foods and are major constituent of coffee, cocoa, tea, cola drinks and chocolate. Although not structurally related, caffeine and caffeic acid has been reported to elicit neuroprotective properties. However, their different proportional distribution in food sources and possible effect of such interactions are not often taken into consideration. Therefore, in this study, we investigated the effect of caffeine, caffeic acid and their various combinations on activities of some enzymes [acetylcholinesterase (AChE), monoamine oxidase (MAO) ecto-nucleoside triphosphate diphosphohydrolase (E-NTPase), ecto-5 1 -nucleotidase (E-NTDase) and Na + /K + ATPase relevant to neurodegeneration in vitro in rat brain. The stock concentration of caffeine and caffiec acid and their various proportional combinations were prepared and their interactions with the activities of these enzymes were assessed (in vitro) in different brain structures. The Fe 2+ and Cu 2+ chelating abilities of the samples were also investigated. The results revealed that caffeine, caffeic acid and their various combinations exhibited inhibitory effect on activities of AChE, MAO, E-NTPase and E-NTDase, but stimulatory effect on Na + /K + ATPase activity. The combinations also exhibited Fe 2+ and Cu 2+ chelating abilities. Considering the various combinations, a higher caffeine to caffeic acid ratio produced significantly highest enzyme modulatory effects; these were significantly lower to the effect of caffeine alone but significantly higher than the effect of caffeic acid alone. These findings may provide new insight into the effect of proportional combination of these bioactive compounds as obtained in many foods especially with respect to their neuroprotective effects. Copyright © 2017 Elsevier B.V. All rights reserved.

PKCε phosphorylates α4β2 nicotinic ACh receptors and promotes recovery from desensitization

PubMed Central

Lee, A M; Wu, D-F; Dadgar, J; Wang, D; McMahon, T; Messing, R O

2015-01-01

Background and Purpose Nicotinic (ACh) receptor recovery from desensitization is modulated by PKC, but the PKC isozymes and the phosphorylation sites involved have not been identified. We investigated whether PKCε phosphorylation of α4β2 nAChRs regulates receptor recovery from desensitization. Experimental Approach Receptor recovery from desensitization was investigated by electrophysiological characterization of human α4β2 nAChRs. Phosphorylation of the α4 nAChR subunit was assessed by immunoblotting of mouse synaptosomes. Hypothermia induced by sazetidine-A and nicotine was measured in Prkce−/− and wild-type mice. Key Results Inhibiting PKCε impaired the magnitude of α4β2 nAChR recovery from desensitization. We identified five putative PKCε phosphorylation sites in the large intracellular loop of the α4 subunit, and mutating four sites to alanines also impaired recovery from desensitization. α4 nAChR subunit phosphorylation was reduced in synaptosomes from Prkce−/− mice. Sazetidine-A-induced hypothermia, which is mediated by α4β2 nAChR desensitization, was more severe and prolonged in Prkce−/− than in wild-type mice. Conclusions and Implications PKCε phosphorylates the α4 nAChR subunit and regulates recovery from receptor desensitization. This study illustrates the importance of phosphorylation in regulating α4β2 receptor function, and suggests that reducing phosphorylation prolongs receptor desensitization and decreases the number of receptors available for activation. PMID:26103136

Cigarette toxin 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces experimental pancreatitis through α7 nicotinic acetylcholine receptors (nAChRs) in mice

PubMed Central

Alahmari, A. A.; Sreekumar, B.; Patel, V.; Ashat, M.; Alexandre, M.; Uduman, A. K.; Akinbiyi, E. O.; Ceplenski, A.; Shugrue, C. A.; Kolodecik, T. R.; Messenger, S. W.; Groblewski, G. E.; Gorelick, F. S.

2018-01-01

Clinical studies have shown that cigarette smoking is a dose-dependent and independent risk factor for acute pancreatitis. Cigarette smoke contains nicotine which can be converted to the potent receptor ligand and toxin, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Previously, we have shown that NNK induces premature activation of pancreatic zymogens in rats, an initiating event in pancreatitis, and this activation is prevented by pharmacologic inhibition of nicotinic acetylcholine receptors (nAChR). In this study, we determined whether NNK mediates pancreatitis through the α7 isoform of nAChR using α7nAChR knockout mice. PCR analysis confirmed expression of non-neuronal α7nAChR in C57BL/6 (WT) mouse and human acinar cells. NNK treatment stimulated trypsinogen activation in acini from WT but not α7nAChR-/- mice. NNK also stimulated trypsinogen activation in human acini. To further confirm these findings, WT and α7nAChR-/- mice were treated with NNK in vivo and markers of pancreatitis were measured. As observed in acini NNK treatment induced trypsinogen activation in WT but not α7nAChR-/- mice. NNK also induced other markers of pancreatitis including pancreatic edema, vacuolization and pyknotic nuclei in WT but not α7nAChR-/- animals. NNK treatment led to increased neutrophil infiltration, a marker of inflammation, in WT mice and to a significantly lesser extent in α7nAChR-/- mice. We also examined downstream targets of α7nAChR activation and found that calcium and PKC activation are involved down stream of NNK stimulation of α7nAChR. In this study we used genetic deletion of the α7nAChR to confirm our previous inhibitor studies that demonstrated NNK stimulates pancreatitis by activating this receptor. Lastly, we demonstrate that NNK can also stimulate zymogen activation in human acinar cells and thus may play a role in human disease. PMID:29870540

Potential anti-cholinesterase and β-site amyloid precursor protein cleaving enzyme 1 inhibitory activities of cornuside and gallotannins from Cornus officinalis fruits.

PubMed

Bhakta, Himanshu Kumar; Park, Chan Hum; Yokozawa, Takako; Tanaka, Takashi; Jung, Hyun Ah; Choi, Jae Sue

2017-07-01

Cholinesterase (ChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors are promising agents for the treatment of Alzheimer's disease (AD). In the present study, we examined the inhibitory activity of seven compounds isolated from the fruits of Cornus officinalis, cornuside, polymeric proanthocyanidins, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, tellimagrandin I, tellimagrandin II, and isoterchebin, against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE1. All of the compounds displayed concentration-dependent in vitro inhibitory activity toward the ChEs and BACE1. Among them, tellimagrandin II exhibited the best inhibitory activity toward ChEs, whereas the best BACE1 inhibitor was 1,2,3,6-tetra-O-galloyl-β-D-glucose. Isoterchebin and polymeric proanthocyanidins were also significant ChE inhibitors. The kinetic and docking studies demonstrated that all compounds interacted with both the catalytic active sites and the peripheral anionic sites of the ChEs and BACE1. Tellimagrandin II, isoterchebin, and the polymeric proanthocyanidins exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. In conclusion, we identified significant ChE and BACE1 inhibitors from Corni Fructus that could have value as new multi-targeted compounds for anti-AD agents.

Does Your Patient's Urine Turns Dark? Alkaptonuria and Low Back Ache: A Literature Review.

PubMed

Kanniyan, Kalaivanan; Pathak, Aditya C; Dhammi, Ish Kumar; Jain, Anil Kumar

2014-01-01

Alkaptonuria is a very rare inborn error of amino acid metabolism due to deficient homogentisic acid (HGA) oxidase enzyme leading to accumulation of HGA in plasma, cartilage, other tissues of human body and its excretion in urine. It has both systemic and peripheral signs and symptoms. Though low back is a common symptom of alkaptonuria but, in the absence of ochronosis it is rare. Alkaptonuria itself is very rare occurrence with no specific treatment option available to reverse the effect as yet. A 38-year-old male, embroidery worker presented with chronic low back ache with history of staining of clothes in infancy. Later on laboratory and the radiological investigation patient was diagnosed to have alkaptonuria without ochronosis. No other systemic manifestation was present. Patient was treated conservatively and responded well. Though alkaptonuria is a very rare disease, and the occurrence of low back-ache in absence of ochronosis is much rarer. One must be aware of this inborn error of metabolism. Early diagnosis though being "diagnosis of exclusion" for low back-ache, high index of suspicion is advantageous as symptomatic treatment of the alkaptonuria can be initiated and evaluation of other systemic organs can be done in early stages itself.

Inhibitory effect of ebselen on cerebral acetylcholinesterase activity in vitro: kinetics and reversibility of inhibition.

PubMed

Martini, Franciele; Bruning, César Augusto; Soares, Suelen Mendonca; Nogueira, Cristina Wayne; Zeni, Gilson

2015-01-01

Ebselen is a synthetic organoselenium compound that has been considered a potential pharmacological agent with low toxicity, showing antioxidant, anti-inflammatory and neuroprotective effects. It is bioavailable, blood-brain barrier permeant and safe based on cellular toxicity and Phase I-III clinical trials. There is evidence that ebselen inhibits acetylcholinesterase (AChE) activity, an enzyme that plays a key role in the cholinergic system by hydrolyzing acetylcholine (ACh), in vitro and ex vivo. This system has a well-known relationship with cognitive process, and AChE inhibitors, such as donepezil and galantamine, have been used to treat cognitive deficits, mainly in the Alzheimer's Disease (AD). However, these drugs have poor bioavailability and a number of side effects, including gastrointestinal upsets and hepatotoxicity. In this way, this study aimed to evaluate the effect of ebselen on cerebral AChE activity in vitro and to determine the kinetic profile and the reversibility of inhibition by dialysis. Ebselen inhibited the cerebral AChE activity with an IC50 of 29 µM, similar to IC50 found with pure AChE from electric eel, demonstrating a mixed and reversible inhibition of AChE, since it increased Km and decreased Vmax. The AChE activity was recovered within 60 min of dialysis. Therefore, the use of ebselen as a therapeutic agent for treatment of AD should be considered, although memory behavior tasks are needed to support such hypothesis.

Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

PubMed

Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki; Duschene, Kaitlin S; Crain, Adam V; Byer, Amanda S; Shepard, Eric M; Rasmussen, Ashley; Yang, Jian; Broderick, William E; Vey, Jessica L; Drennan, Catherine L; Hoffman, Brian M; Broderick, Joan B

2017-08-30

Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.

Crystal structure, phytochemical study and enzyme inhibition activity of Ajaconine and Delectinine

NASA Astrophysics Data System (ADS)

Ahmad, Shujaat; Ahmad, Hanif; Khan, Hidayat Ullah; Shahzad, Adnan; Khan, Ezzat; Ali Shah, Syed Adnan; Ali, Mumtaz; Wadud, Abdul; Ghufran, Mehreen; Naz, Humera; Ahmad, Manzoor

2016-11-01

The Crystal structure, comparative DFT study and phytochemical investigation of atisine type C-20 diterpenoid alkaloid ajaconine (1) and lycoctonine type C-19 diterpenoid alkaloid delectinine (2) is reported here. These compounds were isolated from Delphinium chitralense. Both the natural products 1 and 2 crystallize in orthorhombic crystal system with identical space group of P212121. The geometric parameters of both compounds were calculated with the help of DFT using B3LYP/6-31+G (p) basis set and HOMO-LUMO energies, optimized band gaps, global hardness, ionization potential, electron affinity and global electrophilicity are calculated. The compounds 1 and 2 were screened for acetyl cholinesterase and butyryl cholinesterase inhibition activities in a dose dependent manner followed by molecular docking to explore the possible inhibitory mechanism of ajaconine (1) and delectinine (2). The IC50 values of tested compounds against AChE were observed as 12.61 μM (compound 1) and 5.04 μM (compound 2). The same experiments were performed for inhibition of BChE and IC50 was observed to be 10.18 μM (1) and 9.21 μM (2). Promising inhibition activity was shown by both the compounds against AChE and BChE in comparison with standard drugs available in the market such as allanzanthane and galanthamine. The inhibition efficiency of both the natural products was determined in a dose dependent manner.

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. Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

Changes in acetylcholinesterase, Na+,K+-ATPase, and Mg2+-ATPase activities in the frontal cortex and the hippocampus of hyper- and hypothyroid adult rats.

PubMed

Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Stolakis, Vasileios; Mourouzis, Iordanis; Cokkinos, Dennis; Tsakiris, Stylianos

2007-08-01

The thyroid hormones (THs) are crucial determinants of normal development and metabolism, especially in the central nervous system. The metabolic rate is known to increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na+,K+)- and Mg2+-adenosinetriphosphatase (ATPase) in the frontal cortex and the hippocampus of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, and hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. A region-specific behavior was observed concerning the examined enzyme activities in hyper- and hypothyroidism. In hyperthyroidism, AChE activity was significantly increased only in the hippocampus (+22%), whereas Na+,K+-ATPase activity was significantly decreased in the hyperthyroid rat hippocampus (-47%) and remained unchanged in the frontal cortex. In hypothyroidism, AChE activity was significantly decreased in the frontal cortex (-23%) and increased in the hippocampus (+21%). Na+,K+-ATPase activity was significantly decreased in both the frontal cortex (-35%) and the hippocampus (-43%) of hypothyroid rats. Mg2+-ATPase remained unchanged in the regions of both hyper- and hypothyroid rat brains. Our data revealed that THs affect the examined adult rat brain parameters in a region- and state-specific way. The TH-reduced Na+,K+-ATPase activity may increase the synaptic acetylcholine release and, thus, modulate AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems in the examined brain regions.

Inhibition of acetylcholinesterase activity in the central nervous system of the red swamp crayfish, Procambarus clarkii, by mercury, cadmium, and lead

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devi, M.; Fingerman, M.

The toxicological, physiological and biochemical responses of aquatic crustaceans to heavy metals have been reported by several investigators. Levels of glucose, lactic acid, sodium, potassium, aspartate aminotransferase and alanine aminotransferase in the blood of the crab Scylla serrata increased, while glycogen levels in hepatopancreas and muscle decreased after a four-week exposure to mercuric chloride. In fiddler crab, Uca pugilator, enzyme activity was observed to decrease in the hepatopancreas but increased in abdominal muscle after 48 hr cadmium exposure. In the red swamp crayfish, Procambarus clarkii, exposed for 96 hr to cadmium, glutahione (GSH) level and GSH S-transferase activity deceased inmore » the midgut. In crayfish Astacus astacus exposed to sublethal concentrations of lead and cadmium, oxidative enzyme (succine dehydrogenase and NADPH-cytochrome P450 reductase) activities in gills and hepatopancrease decreased. Acetylcholinesterase (AChE) inhibition by organophosphates and organocarbamates in various crustaceans has bee reported. In vivo cadmium exposure caused increases in esterase activities, but mercury exposure decreases these activities in the hepatopancreas of the shrimp Callianassa tyrrhena. The freshwater crab, Barytelphusa guerini, exposed to 0.6 ppm cadmium showed reduced oxygen consumption throughout the experiment whereas AChE activity increased after 4 days but decreased after 15 days. The authors wanted to determine the effects of cadmium, lead and mercury on AChE activity in central nervous tissue of Procambarus clarkii. This enzyme has the potential for serving both as a biochemical indicator of toxic stress and a sensitive parameter for testing water for the presence of toxicants. These three biologically silent metals have, according to Schweinsberg and Karsa great toxicological significance to humans because their use is widespread. 14 refs., 4 figs.« less

Cholinesterase inhibitory activities of Apai-sa-le recipe and its ingredients.

PubMed

Senavong, Pimolvan; Sattaponpan, Chitsanucha; Silavat Suk-um; Itharat, Arunporn

2014-08-01

Acetylcholinesterase and butyrylcholoinesterase inhibitors are well-known drugs commonly used in the treatment ofAlzheimer's disease (AD) to improve cognitive function. These enzyme inhibitors were reported to be found in manyplants. Apai-sa-le recipe was a Thai tradition used as nootropic recipe and formerly claimed to improve memory. Therefore, it is interesting to investigate cholinesterase inhibitory activity ofthe recipe and its ingredients. To determine the whole recipe ofApai-sa-le and its ingredients for inhibitory effect on acetylcholinesterase (AChE) and human butyrylcholinesterase (BuChE) activities. Thirty grams of each plant and 181 grams of the whole recipe were separately extracted by 95% ethanol, after filtered the filtrate were evaporated and vacuum-dried at 45°C. By Elman method, the inhibitory activities of both enzymes were assessed. The volatile constituents ofeach extract were determined by GCMS. The constituents in the non- volatile extract were examined by TLC and the antioxidant activity was determined. Four plants exhibited specific BuChE inhibitor were Lepidium sativum Linn. (Ls), Piper nigrum L. (Pn), Angelica dahurica Benth (Ad) andAtractylodes lancea DC. (Al), which shown the lC50 of 5.59, 24.52, 73.23, 96.25 μg/ml, respectively whereas galantamine and the whole recipe showed IC50 of 0.59 and 236 μg/ml. Only Pn extract inhibited AChE at lCso of 25.46 μg/ml. By GCMS and TLC fingerprints revealed the main constituents in LS, Ad, Al andPn as apiol, cumialdehyde, furanodiene and piperine. Moreover nine plant extracts and the whole recipe showed antioxidant activity. Lepidium sativum Linn. (Ls) extract showed the most potency on BuChE inhibitory effect. Three ingredients and the whole recipe exhibited mild activity. Only Piper nigrum L demonstrated inhibition effect on both AChE and BuChE.

Chitosan-coated polystyrene microplate for covalent immobilization of enzyme.

PubMed

Zhang, Yaodong; Li, Li; Yu, Caihong; Hei, Tingting

2011-10-01

Microplates made of polystyrene have been widely used for immunoassays. Protein molecules that have been immobilized on a hydrophobic polystyrene microplate by passive adsorption lose their activity and suffer considerable denaturation. A new chitosan-coated microplate suitable for the covalent immobilization of enzymes has been developed. The primary amino groups of chitosan were exploited for this covalent coupling of proteins. The optical transmittance of the chitosan-coated microplate, at wavelengths of 400-800 nm, was estimated to be suitable for its application in chromogenic reaction-based bioassays. The immobilization efficiency of the chitosan-coated microplate was demonstrated to be far superior to that of a conventional microplate when tested using acetylcholinesterase (AChE) and β-glucosidase as model biomolecules, and the chitosan-coated microplate may thus have potential applications in biosensing and bioreactor systems. © Springer-Verlag 2011

Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells.

PubMed

Qian, Jie; Mummalaneni, Shobha K; Alkahtani, Reem M; Mahavadi, Sunila; Murthy, Karnam S; Grider, John R; Lyall, Vijay

2016-01-01

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and β nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-β-erythroidine, a α4β2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and β4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

Regulation of ACh release from guinea pig bladder urothelial cells: potential role in bladder filling sensations.

PubMed

McLatchie, L M; Young, J S; Fry, C H

2014-07-01

The aim of this study was to quantify and characterize the mechanism of non-neuronal ACh release from bladder urothelial cells and to determine if urothelial cells could be a site of action of anti-muscarinic drugs. A novel technique was developed whereby ACh could be measured from freshly isolated guinea pig urothelial cells in suspension following mechanical stimulation. Various agents were used to manipulate possible ACh release pathways in turn and to study the effects of muscarinic receptor activation and inhibition on urothelial ATP release. Minimal mechanical stimulus achieved full ACh release, indicating a small dynamic range and possible all-or-none signal. ACh release involved a mechanism dependent on the anion channel CFTR and intracellular calcium concentration, but was independent of extracellular calcium, vesicular trafficking, connexins or pannexins, organic cation transporters and was not affected by botulinum-A toxin. Stimulating ACh receptors increased ATP production and antagonizing them reduced ATP release, suggesting a link between ACh and ATP release. These results suggest that release of non-neuronal ACh from the urothelium is large enough and well located to act as a modulator of ATP release. It is hypothesized that this pathway may contribute to the actions of anti-muscarinic drugs in reducing the symptoms of lower urinary tract syndromes. Additionally the involvement of CFTR in ACh release suggests an exciting new direction for the treatment of these conditions. © 2014 The British Pharmacological Society.

Endogenous purines modulate K+ -evoked ACh secretion at the mouse neuromuscular junction.

PubMed

Guarracino, Juan F; Cinalli, Alejandro R; Veggetti, Mariela I; Losavio, Adriana S

2018-06-01

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y 13 and A 1 , A 2A , and A 3 receptors, respectively. To elucidate the action of endogenous purines on K + -dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end-plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K + , the P2Y 13 antagonist N-[2-(methylthio)ethyl]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR-C69931MX) increased asynchronous ACh secretion while the A 1 , A 3 , and A 2A antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(±)-dihydropyridine-3,5-, dicarboxylate (MRS-1191), and 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S-(p-nitrobenzyl)-6-thioinosine provoked a reduction of 10 mM K + -evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K + , endogenous ATP/ADP and adenosine bind to inhibitory P2Y 13 and A 1 and A 3 receptors since AR-C69931MX, DPCPX, and MRS-1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto-5'-nucleotidase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K + , suggesting that more adenosine is needed to activate excitatory A 2A receptors. At high K + concentration, the equilibrative transporters appear to be saturated allowing the accumulation of

Does Your Patient’s Urine Turns Dark? Alkaptonuria and Low Back Ache: A Literature Review

PubMed Central

Kanniyan, Kalaivanan; Pathak, Aditya C; Dhammi, Ish Kumar; Jain, Anil Kumar

2014-01-01

Introduction: Alkaptonuria is a very rare inborn error of amino acid metabolism due to deficient homogentisic acid (HGA) oxidase enzyme leading to accumulation of HGA in plasma, cartilage, other tissues of human body and its excretion in urine. It has both systemic and peripheral signs and symptoms. Though low back is a common symptom of alkaptonuria but, in the absence of ochronosis it is rare. Alkaptonuria itself is very rare occurrence with no specific treatment option available to reverse the effect as yet. Case Report: A 38-year-old male, embroidery worker presented with chronic low back ache with history of staining of clothes in infancy. Later on laboratory and the radiological investigation patient was diagnosed to have alkaptonuria without ochronosis. No other systemic manifestation was present. Patient was treated conservatively and responded well. Conclusion: Though alkaptonuria is a very rare disease, and the occurrence of low back-ache in absence of ochronosis is much rarer. One must be aware of this inborn error of metabolism. Early diagnosis though being “diagnosis of exclusion” for low back-ache, high index of suspicion is advantageous as symptomatic treatment of the alkaptonuria can be initiated and evaluation of other systemic organs can be done in early stages itself. PMID:27298997

Inhibition of neutral endopeptidase increases airway responsiveness to ACh in nonsensitized normal rats.

PubMed

Chiba, Y; Misawa, M

1995-02-01

The effects of sensory neuropeptides on the airway responsiveness to acetylcholine (ACh) were investigated in normal nonsensitized rats. The airway responsiveness to inhaled ACh was significantly increased after treatment with neurokinin A (NKA; 0.001%) or substance P (SP; 0.01%) aerosol in the presence of the neutral endopeptidase (NEP) inhibitor. NKA had a more potent effect than SP. Interestingly, the intravenous treatment with NEP inhibitor alone also induced airway hyperresponsiveness (AHR) to inhaled ACh. This AHR was significantly attenuated by pretreatment with a nonselective NK-receptor antagonist, [D-Pro2,D-Trp7,9]SP, systemic capsaicin, or bilateral cervical vagotomy, indicating that decreased NEP activity results in accumulation of endogenous sensory neuropeptide(s) and enhancement of vagal reflex to cause AHR. The airway responsiveness to ACh of isolated left main bronchus was also increased after treatment with 10(-6) M NKA, but not SP, together with 10(-6) M phosphoramidon. This in vitro AHR to ACh induced by phosphoramidon plus NKA was significantly attenuated by pretreatment with 10(-6) M tetrodotoxin. These findings suggest that overaccumulated sensory neuropeptides, especially NKA, may enhance the probability of transmitter release, probably via NK2 receptors, and that the enhanced transmitter release might be involved in AHR in rats.

Synthesis, spectroscopic, computational (DFT/B3LYP), AChE inhibition and antioxidant studies of imidazole derivative

NASA Astrophysics Data System (ADS)

Ahmad, Faheem; Alam, Mohammad Jane; Alam, Mahboob; Azaz, Shaista; Parveen, Mehtab; Park, Soonheum; Ahmad, Shabbir

2018-01-01

The present study reports the synthesis and evaluation of biological properties of 3a,8a-dihydroxy-8-oxo-1,3,3a,8a-tetrahydroindeno[1,2-d]imidazol-2(1H)-iminium chloride (3). The structure was confirmed by the FTIR, NMR, MS, CHN microanalysis and X-ray crystallographic analysis. Quantum chemical calculations have been performed at B3LYP-D3/6-311++G(d,p) level of theory to study the molecular geometry, IR, (1H and 13C) NMR, UV/Vis spectra and other molecular parameters of the asymmetric unit of crystal of imidazole compound (3). An empirical dispersion correction to hybrid functional (B3LYP-D3) has been incorporated in the present calculations due to presence of non-covalent interaction, Cl⋯H-O, in the present compound. The remarkable agreement has been observed between theoretical data and those measured experimentally. Moreover, the Hirshfeld analysis was carried out to ascertain the secondary interactions and associated 2D fingerprint plots. The synthesized imidazole derivative showed promising antioxidant property and inhibitory activity against acetylcholinesterase (AChE). Molecular docking was also performed in order to explain in silico antioxidant studies and to ascertain the probable binding mode of compound with the amino acid residues of protein.

Nicotinic ACh Receptors as Therapeutic Targets in CNS Disorders

PubMed Central

Dineley, Kelly T.; Pandya, Anshul A.; Yakel, Jerrel L.

2015-01-01

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor channels (nAChRs). These receptors are widely distributed throughout the central nervous system, being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in the mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer’s disease), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain. PMID:25639674

Nicotinic ACh receptors as therapeutic targets in CNS disorders.

PubMed

Dineley, Kelly T; Pandya, Anshul A; Yakel, Jerrel L

2015-02-01

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor (nAChR) channels. These receptors are widely distributed throughout the central nervous system (CNS), being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however, the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer's disease, AD), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain. Published by Elsevier Ltd.

Structure-activity relationship investigation of tertiary amine derivatives of cinnamic acid as acetylcholinesterase and butyrylcholinesterase inhibitors: compared with that of phenylpropionic acid, sorbic acid and hexanoic acid.

PubMed

Gao, Xiaohui; Tang, Jingjing; Liu, Haoran; Liu, Linbo; Kang, Lu; Chen, Wen

2018-12-01

In the present investigation, 48 new tertiary amine derivatives of cinnamic acid, phenylpropionic acid, sorbic acid and hexanoic acid (4d-6g, 10d-12g, 16d-18g and 22d-24g) were designed, synthesized and evaluated for the effect on AChE and BChE in vitro. The results revealed that the alteration of aminoalkyl types and substituted positions markedly influences the effects in inhibiting AChE. Almost of all cinnamic acid derivatives had the most potent inhibitory activity than that of other acid derivatives with the same aminoalkyl side chain. Unsaturated bond and benzene ring in cinnamic acid scaffold seems important for the inhibitory activity against AChE. Among them, compound 6g revealed the most potent AChE inhibitory activity (IC 50 value: 3.64 µmol/L) and highest selectivity over BChE (ratio: 28.6). Enzyme kinetic study showed that it present a mixed-type inhibition against AChE. The molecular docking study suggested that it can bind with the catalytic site and peripheral site of AChE.

Effect of diet on carboxylesterase activity of tadpoles (Rhinella arenarum) exposed to chlorpyrifos.

PubMed

Attademo, A M; Sanchez-Hernandez, J C; Lajmanovich, R C; Peltzer, P M; Junges, C

2017-01-01

An outdoor microcosm was performed with tadpoles (Rhinella arenarum) exposed to 125μgL -1 chlorpyrifos and fed two types of food, i.e., lettuce (Lactuca sativa) and a formulated commercial pellet. Acetylcholinesterase (AChE) and carboxylesterase (CbE) activities were measured in liver and intestine after 10 days of pesticide exposure. Non-exposed tadpoles fed lettuce had an intestinal AChE activity almost two-fold higher than that of pellet-fed tadpoles. No significant differences were observed, however, in liver AChE activity between diets. Likewise, intestinal CbE activity - measured using two substrates, i.e. 1-naphthyl acetate (1-NA) and 4-nitrophenyl valerate (4-NPV) - was higher in tadpoles fed lettuce than in those fed pellets. However, the diet-dependent response of liver CbE activity was opposite to that in the intestine. Chlorpyrifos caused a significant inhibition of both esterase activities, which was tissue- and diet-specific. The highest inhibition degree was found in the intestinal AChE and CbE activities of lettuce-fed tadpoles (42-78% of controls) compared with pellet-fed tadpoles (<60%). Although chlorpyrifos significantly inhibited liver CbE activity of the group fed lettuce, this effect was not observed in the group fed pellets. In general, intestinal CbE activity was more sensitive to chlorpyrifos inhibition than AChE activity. This finding, together with the high levels of basal CbE activity found in the intestine, may be understood as a detoxification system able to reduce intestinal OP uptake. Moreover, the results of this study suggest that diet is a determinant factor in toxicity testing with tadpoles to assess OP toxicity, because it modulates levels of this potential detoxifying enzyme activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition of cholinesterase activity by extracts, fractions and compounds from Calceolaria talcana and C. integrifolia (Calceolariaceae: Scrophulariaceae).

PubMed

Cespedes, Carlos L; Muñoz, Evelyn; Salazar, Juan R; Yamaguchi, Lydia; Werner, Enrique; Alarcon, Julio; Kubo, Isao

2013-12-01

Extracts, fractions and compounds from Calceolaria talcana and C. integrifolia exhibited strong inhibitory effects of the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the in vitro Ellman's method. The most active samples were from the ethyl acetate extract, which caused a mixed-type inhibition against AChE (69.8% and 79.5% at 100 and 200 μg/ml, respectively) and against BChE (98.5% and 99.8% at 100 and 200 μg/ml, respectively) and its major components verbascoside 8 (50.9% and 70.0% at 200 μg/ml, against AChE and BChE, respectively), martynoside 9, and fraction F-7 (which corresponds to a mixture of 8, 9, and other phenylethanoids and phenolics that remain unidentified) (80.2% and 85.3% at 100 and 200 μg/ml, against AChE, respectively and 99.1% and 99.7% at 100 and 200 μg/ml, against BChE, respectively) inhibited the acetylcholinesterase enzyme competitively. The most polar fraction F-5 from n-hexane extract (a mixture of naphthoquinones: 2-hydroxy-3-(1,1-dimethylallyl-1,4-naphthoquinone) 6, α-dunnione 7 and other polar compounds that remain unidentified) showed a mixed-type inhibition (71.5% and 72.1% against AChE and BChE at 200 μg/ml, respectively). Finally, the methanol-soluble residue presented a complex, mixed-type inhibition (39.9% and 67.9% against AChE and BChE at 200 μg/ml, respectively). The mixture F-3 with diterpenes was obtained from the n-hexane extract: (1,10-cyclopropyl-9-epi-ent-isopimarol) 1, 19-α-hydroxy-abietatriene 2, and F-4 a mixture of triterpenes α-lupeol 3, β-sitosterol 4, ursolic acid 5 together with a complex mixture of terpenes that did not show activity. In summary, extracts and natural compounds from C. talcana and C. integrifolia were isolated, identified and characterized as cholinesterase inhibitors.

Ionic liquid mediated synthesis and molecular docking study of novel aromatic embedded Schiff bases as potent cholinesterase inhibitors.

PubMed

Abd Razik, Basma M; Osman, Hasnah; Basiri, Alireza; Salhin, Abdussalam; Kia, Yalda; Ezzat, Mohammed Oday; Murugaiyah, Vikneswaran

2014-12-01

Novel aromatic embedded Schiff bases have been synthesized in ionic liquid [bmim]Br and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes inhibitory activities. Among the newly synthesized compounds, 5f, 5h and 7j displayed higher AChE enzyme inhibitory activities than standard drug, galanthamine, with IC50 values of 1.88, 2.05 and 2.03μM, respectively. Interestingly, all the compounds except for compound 5c displayed higher BChE inhibitories than standard with IC50 values ranging from 3.49 to 19.86μM. Molecular docking analysis for 5f and 7j possessing the most potent AChE and BChE inhibitory activities, disclosed their binding interaction templates to the active site of AChE and BChE enzymes, respectively. Copyright © 2014 Elsevier Inc. All rights reserved.

Increased serum butyrylcholinesterase activity in type IIb hyperlipidaemic patients.

PubMed

Kálmán, János; Juhász, Anna; Rakonczay, Zoltán; Abrahám, György; Zana, Marianna; Boda, Krisztina; Farkas, Tibor; Penke, Botond; Janka, Zoltán

2004-07-23

The inheritance of the apolipoprotein E4 (APOE4) allele has been shown to increase the plasma cholesterol level, but little information is as concerns the association of the APOE genotype and hyperlipidaemia and the activities of two serum enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Blood samples from 55 type IIb hyperlipidaemic, non-demented patients and 55 age- and sex-matched controls were therefore examined in this pilot study. A significantly increased BChE activity was found in the serum of type IIb hyperlipidaemic patients, but the AChE activity did not differ significantly as compared with that in the control group. The APOE4 allele was significantly overrepresented among the hyperlipidaemic probands, but neither serum cholinesterase activity was affected by the dosage of the APOE4 gene. Our results point to a possible association between an abnormal lipid metabolism and the BChE activity and might have implications as regards the pathomechanism of both Alzheimer's and vascular dementias and the cholinesterase inhibitor therapy of dementing disorders.

Immune responses to HTLV-I(ACH) during acute infection of pig-tailed macaques.

PubMed

McGinn, Therese M; Wei, Qing; Stallworth, Jackie; Fultz, Patricia N

2004-04-01

Human T cell lymphotropic virus type 1 (HTLV-I) is causally linked to adult T cell leukemia/lymphoma (ATL) and a chronic progressive neurological disease, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A nonhuman primate model that reproduces disease symptoms seen in HTLV-I-infected humans might facilitate identification of initial immune responses to the virus and an understanding of pathogenic mechanisms in HTLV-I-related disease. Previously, we showed that infection of pig-tailed macaques with HTLV-I(ACH) is associated with multiple signs of disease characteristic of both HAM/TSP and ATL. We report here that within the first few weeks after HTLV-I(ACH) infection of pig-tailed macaques, serum concentrations of interferon (IFN)-alpha increased and interleukin-12 decreased transiently, levels of nitric oxide were elevated, and activation of CD4(+) and CD8(+) lymphocytes and CD16(+) natural killer cells in peripheral blood were observed. HTLV-I(ACH) infection elicited virus-specific antibodies in all four animals within 4 to 6 weeks; however, Tax-specific lymphoproliferative responses were not detected until 25-29 weeks after infection in all four macaques. IFN-gamma production by peripheral blood cells stimulated with a Tax or Gag peptide was detected to varying degrees in all four animals by ELISPOT assay. Peripheral blood lymphocytes from one animal that developed only a marginal antigen-specific cellular response were unresponsive to mitogen stimulation during the last few weeks preceding its death from a rapidly progressive disease syndrome associated with HTLV-I(ACH) infection of pig-tailed macaques. The results show that during the first few months after HTLV-I(ACH) infection, activation of both innate and adaptive immunity, limited virus-specific cellular responses, sustained immune system activation, and, in some cases, immunodeficiency were evident. Thus, this animal model might be valuable for understanding early stages of infection

AChR-specific immunosuppressive therapy of myasthenia gravis.

PubMed

Luo, Jie; Lindstrom, Jon

2015-10-15

Myasthenia gravis (MG) is an organ-specific autoimmune disease characterized by muscle fatigability. In most cases, it is mediated by autoantibodies targeting muscle nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. Experimental autoimmune myasthenia gravis (EAMG) is an animal model for MG, which is usually induced by immunization with AChR purified from fish electric organ. Pathological autoantibodies to AChRs are directed at the extracellular surface, especially the main immunogenic region (MIR). Current treatments for MG can help many but not all patients. Antigen-specific immunosuppressive therapy for MG that specifically suppresses the autoimmune response without affecting the entire immune system and avoids side effects of general immunosuppression is currently unavailable. Early attempts at antigen-specific immunosuppression for EAMG using AChR extracellular domain sequences that form epitopes for pathological autoantibodies risked provoking autoimmunity rather than suppressing it. We discovered a novel approach to specific immunosuppression of EAMG with a therapeutic vaccine consisting of bacterially-expressed human AChR cytoplasmic domains, which has the potential to specifically suppress MG without danger of causing exacerbation. This approach prevents development of chronic EAMG when initiated immediately after the acute phase of EAMG, and rapidly reverses established chronic EAMG when started during the chronic phase of EAMG. Successfully treated rats exhibited long-term resistance to re-induction of EAMG. In this review we also discuss the current understanding of the mechanisms by which the therapy works. Vaccination with AChR cytoplasmic domains in adjuvant is promising as a safe, antigen-specific, potent, effective, rapidly acting, and long lasting approach to therapy of MG. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

PubMed

Tillman, Tommy S; Alvarez, Frances J D; Reinert, Nathan J; Liu, Chuang; Wang, Dawei; Xu, Yan; Xiao, Kunhong; Zhang, Peijun; Tang, Pei

2016-08-26

Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Droplet-based microfluidics for dose-response assay of enzyme inhibitors by electrochemical method.

PubMed

Gu, Shuqing; Lu, Youlan; Ding, Yaping; Li, Li; Zhang, Fenfen; Wu, Qingsheng

2013-09-24

A simple but robust droplet-based microfluidic system was developed for dose-response enzyme inhibition assay by combining concentration gradient generation method with electrochemical detection method. A slotted-vials array and a tapered tip capillary were used for reagents introduction and concentration gradient generation, and a polydimethylsiloxane (PDMS) microfluidic chip integrated with microelectrodes was used for droplet generation and electrochemical detection. Effects of oil flow rate and surfactant on electrochemical sensing were investigated. This system was validated by measuring dose-response curves of three types of acetylcholinesterase (AChE) inhibitors, including carbamate pesticide, organophosphorus pesticide, and therapeutic drugs regulating Alzheimer's disease. Carbaryl, chlorpyrifos, and tacrine were used as model analytes, respectively, and their IC50 (half maximal inhibitory concentration) values were determined. A whole enzyme inhibition assay was completed in 6 min, and the total consumption of reagents was less than 5 μL. This microfluidic system is applicable to many biochemical reactions, such as drug screening and kinetic studies, as long as one of the reactants or products is electrochemically active. Copyright © 2013 Elsevier B.V. All rights reserved.

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

PubMed

Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

2003-05-13

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

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

PubMed Central

Wheelock, Craig E.; Eder, Kai J.; Werner, Inge; Huang, Huazhang; Jones, Paul D.; Brammell, Benjamin F.; Elskus, Adria A.; Hammock, Bruce D.

2006-01-01

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

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

USGS Publications Warehouse

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

2005-01-01

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

Monoamine uptake inhibitors block alpha7-nAChR-mediated cerebral nitrergic neurogenic vasodilation.

PubMed

Long, Cheng; Chen, Mei-Fang; Sarwinski, Susan J; Chen, Po-Yi; Si, Minliang; Hoffer, Barry J; Evans, M Steven; Lee, Tony J F

2006-07-01

We have proposed that activation of cerebral perivascular sympathetic alpha7-nicotinic acetylcholine receptors (alpha7-nAChRs) by nicotinic agonists releases norepinephrine, which then acts on parasympathetic nitrergic nerves, resulting in release of nitric oxide and vasodilation. Using patch-clamp electrophysiology, immunohistochemistry, and in vitro tissue bath myography, we tested this axo-axonal interaction hypothesis further by examining whether blocking norepinephrine reuptake enhanced alpha7-nAChR-mediated cerebral nitrergic neurogenic vasodilation. The results indicated that choline- and nicotine-induced alpha7-nAChR-mediated nitrergic neurogenic relaxation in endothelium-denuded isolated porcine basilar artery rings was enhanced by desipramine and imipramine at lower concentrations (0.03-0.1 microM) but inhibited at higher concentrations (0.3-10 microM). In cultured superior cervical ganglion (SCG) neurons of the pig and rat, choline (0.1-30 mM)-evoked inward currents were reversibly blocked by 1-30 microM mecamylamine, 1-30 microM methyllycaconitine, 10-300 nM alpha-bungarotoxin, and 0.1-10 microM desipramine and imipramine, providing electrophysiological evidence for the presence of similar functional alpha7-nAChRs in cerebral perivascular sympathetic neurons of pigs and rats. In alpha7-nAChR-expressing Xenopus oocytes, choline-elicited inward currents were attenuated by alpha-bungarotoxin, imipramine, and desipramine. These monoamine uptake inhibitors appeared to directly block the alpha7-nAChR, resulting in diminished nicotinic agonist-induced cerebral nitrergic vasodilation. The enhanced nitrergic vasodilation by lower concentrations of monoamine uptake inhibitors is likely due to a greater effect on monoamine uptake than on alpha7-nAChR blockade. These results further support the hypothesis of axo-axonal interaction in nitrergic regulation of cerebral vascular tone.

County-scale spatial distribution of soil enzyme activities and enzyme activity indices in agricultural land: implications for soil quality assessment.

PubMed

Tan, Xiangping; Xie, Baoni; Wang, Junxing; He, Wenxiang; Wang, Xudong; Wei, Gehong

2014-01-01

Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km(2)) scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI) and the geometric mean of enzyme activities (GME). At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality.

RgIA4 Potently Blocks Mouse α9α10 nAChRs and Provides Long Lasting Protection against Oxaliplatin-Induced Cold Allodynia.

PubMed

Christensen, Sean B; Hone, Arik J; Roux, Isabelle; Kniazeff, Julie; Pin, Jean-Philippe; Upert, Grégory; Servent, Denis; Glowatzki, Elisabeth; McIntosh, J Michael

2017-01-01

Transcripts for α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are found in diverse tissues. The function of α9α10 nAChRs is best known in mechanosensory cochlear hair cells, but elsewhere their roles are less well-understood. α9α10 nAChRs have been implicated as analgesic targets and α-conotoxins that block α9α10 nAChRs produce analgesia. However, some of these peptides show large potency differences between species. Additionally several studies have indicated that these conotoxins may also activate GABA B receptors (GABA B Rs). To further address these issues, we cloned the cDNAs of mouse α9 and α10 nAChR subunits. When heterologously expressed in Xenopus oocytes, the resulting α9α10 nAChRs had the expected pharmacology of being activated by acetylcholine and choline but not by nicotine. A conotoxin analog, RgIA4, potently, and selectively blocked mouse α9α10 nAChRs with low nanomolar affinity indicating that RgIA4 may be effectively used to study murine α9α10 nAChR function. Previous reports indicated that RgIA4 attenuates chemotherapy-induced cold allodynia. Here we demonstrate that RgIA4 analgesic effects following oxaliplatin treatment are sustained for 21 days after last RgIA4 administration indicating that RgIA4 may provide enduring protection against nerve damage. RgIA4 lacks activity at GABA B receptors; a bioluminescence resonance energy transfer assay was used to demonstrate that two other analgesic α-conotoxins, Vc1.1 and AuIB, also do not activate GABA B Rs expressed in HEK cells. Together these findings further support the targeting of α9α10 nAChRs in the treatment of pain.

Aflatoxins produced by Aspergillus parasiticus present in the diet of quails increase the activities of cholinesterase and adenosine deaminase.

PubMed

da Silva, Aleksandro Schafer; Santurio, Janio M; Roza, Lenilson F; Bottari, Nathieli B; Galli, Gabriela M; Morsch, Vera M; Schetinger, Maria Rosa C; Baldissera, Matheus D; Stefani, Lenita M; Radavelli, Willian M; Tomasi, Thainã; Boiago, Marcel M

2017-06-01

The aim of this study was to evaluate the effects of aflatoxins on cholinesterases (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and adenosine deaminase (ADA) activities in quails. For this, twenty male quails were randomly distributed into two groups (n = 10 each): the group A was composed by quails that received feed without aflatoxin (the control group); while the group B was composed by quails that received feed contaminated with 200 ppm/kg of feed of aflatoxin. On day 20, the animals were euthanized to measure the activities of AChE (total blood and brain), BChE (serum) and ADA (serum, liver, and brain), as well as for histopathological analyses (liver and intestine). AChE, BChE, and ADA levels increased in animals intoxicated by aflatoxin compared to the control group. The presence of aflatoxin lead to severe hydropic degeneration of hepatocytes and small focus of hepatocyte necrosis. In conclusion, aflatoxins poisoning increased AChE, BChE, and ADA activities, suggesting the involvement of these enzymes during this type of intoxication, in addition to the fact that they are well known molecules that participate in physiological and pathological events as inflammatory mediators. In summary, increased AChE, BChE and ADA activities contribute directly to the inflammatory process and tissue damage, and they might be involved in disease development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization.

PubMed

Frobert, Y; Créminon, C; Cousin, X; Rémy, M H; Chatel, J M; Bon, S; Bon, C; Grassi, J

1997-05-23

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and

Acetylcholine-induced seizure-like activity and modified cholinergic gene expression in chronically epileptic rats.

PubMed

Zimmerman, Gabriel; Njunting, Marleisje; Ivens, Sebastian; Tolner, Else A; Tolner, Elsa; Behrens, Christoph J; Gross, Miriam; Soreq, Hermona; Heinemann, Uwe; Friedman, Alon

2008-02-01

The entorhinal cortex (EC) plays an important role in temporal lobe epilepsy. Under normal conditions, the enriched cholinergic innervation of the EC modulates local synchronized oscillatory activity; however, its role in epilepsy is unknown. Enhanced neuronal activation has been shown to induce transcriptional changes of key cholinergic genes and thus alter cholinergic responses. To examine cholinergic modulations in epileptic tissue we studied molecular and electrophysiological cholinergic responses in the EC of chronically epileptic rats following exposure to pilocarpine or kainic acid. We confirmed that while the total activity of the acetylcholine (ACh)-hydrolysing enzyme, acetylcholinesterase (AChE) was not altered, epileptic rats showed alternative splicing of AChE pre-mRNA transcripts, accompanied by a shift from membrane-bound AChE tetramers to soluble monomers. This was associated with increased sensitivity to ACh application: thus, in control rats, ACh (10-100 microm) induced slow (< 1Hz), periodic events confined to the EC; however, in epileptic rats, ACh evoked seconds-long seizure-like events with initial appearance in the EC, and frequent propagation to neighbouring cortical regions. ACh-induced seizure-like events could be completely blocked by the non-specific muscarinic antagonist, atropine, and were partially blocked by the muscarinic-1 receptor antagonist, pirenzepine; but were not affected by the non-specific nicotinic antagonist, mecamylamine. Epileptic rats presented reduced transcript levels of muscarinic receptors with no evidence of mRNA editing or altered mRNA levels for nicotinic ACh receptors. Our findings suggest that altered cholinergic modulation may initiate seizure events in the epileptic temporal cortex.

Gold nanoclusters-Cu(2+) ensemble-based fluorescence turn-on and real-time assay for acetylcholinesterase activity and inhibitor screening.

PubMed

Sun, Jian; Yang, Xiurong

2015-12-15

Based on the specific binding of Cu(2+) ions to the 11-mercaptoundecanoic acid (11-MUA)-protected AuNCs with intense orange-red emission, we have proposed and constructed a novel fluorescent nanomaterials-metal ions ensemble at a nonfluorescence off-state. Subsequently, an AuNCs@11-MUA-Cu(2+) ensemble-based fluorescent chemosensor, which is amenable to convenient, sensitive, selective, turn-on and real-time assay of acetylcholinesterase (AChE), could be developed by using acetylthiocholine (ATCh) as the substrate. Herein, the sensing ensemble solution exhibits a marvelous fluorescent enhancement in the presence of AChE and ATCh, where AChE hydrolyzes its active substrate ATCh into thiocholine (TCh), and then TCh captures Cu(2+) from the ensemble, accompanied by the conversion from fluorescence off-state to on-state of the AuNCs. The AChE activity could be detected less than 0.05 mU/mL within a good linear range from 0.05 to 2.5 mU/mL. Our proposed fluorescence assay can be utilized to evaluate the AChE activity quantitatively in real biological sample, and furthermore to screen the inhibitor of AChE. As far as we know, the present study has reported the first analytical proposal for sensing AChE activity in real time by using a fluorescent nanomaterials-Cu(2+) ensemble or focusing on the Cu(2+)-triggered fluorescence quenching/recovery. This strategy paves a new avenue for exploring the biosensing applications of fluorescent AuNCs, and presents the prospect of AuNCs@11-MUA-Cu(2+) ensemble as versatile enzyme activity assay platforms by means of other appropriate substrates/analytes. Copyright © 2015 Elsevier B.V. All rights reserved.

Are linear AChR epitopes the real culprit in ocular myasthenia gravis?

PubMed

Wu, Xiaorong; Tüzün, Erdem

2017-02-01

Extraocular muscle weakness occurs in most of the myasthenia gravis (MG) patients and it is often the initial complaint. Approximately 10-20% of MG patients with extraocular muscle weakness display only ocular symptoms and rest of the patients subsequently develop generalized muscle weakness. It is not entirely clear why some MG patients develop only ocular symptoms and why extraocular muscle weakness almost always precedes generalized muscle weakness. These facts are often explained by increased susceptibility of extraocular muscles due to their reduced endplate safety factor and lower complement inhibitor expression. Findings of a recently developed animal model of ocular MG suggest that additional factors might be in play. While immunization of HLA transgenic and wild-type (WT) mice with the native acetylcholine receptor (AChR) pentamer carrying conformational epitopes generates severe generalized muscle weakness, immunization of the same mouse strains with recombinant unfolded AChR subunits containing linear epitopes induces ptosis with or without mild generalized muscle weakness. Notably, immunization of mice with deficient T helper cell-mediated antigen presentation with recombinant AChR subunits or whole native AChR pentamer also induces ocular symptoms, AChR-reactive B cells and AChR antibodies. Based on these findings, we hypothesize that ocular symptoms observed in the earlier stages of MG might be triggered by linear and non-conformational AChR epitopes expressed by thymic cells or invading microorganisms. This initial AChR autoimmunity might be managed by T cell-independent and B cell mediated mechanisms yielding low affinity AChR antibodies. These antibodies are putatively capable of inducing muscle weakness only in extraocular muscles which have increased vulnerability due to their inherent biological properties. After this initial attack, as AChR bearing immune complexes form and the immune system gains access to the native AChR expressed by muscle

Design and prediction of new acetylcholinesterase inhibitor via quantitative structure activity relationship of huprines derivatives.

PubMed

Zhang, Shuqun; Hou, Bo; Yang, Huaiyu; Zuo, Zhili

2016-05-01

Acetylcholinesterase (AChE) is an important enzyme in the pathogenesis of Alzheimer's disease (AD). Comparative quantitative structure-activity relationship (QSAR) analyses on some huprines inhibitors against AChE were carried out using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and hologram QSAR (HQSAR) methods. Three highly predictive QSAR models were constructed successfully based on the training set. The CoMFA, CoMSIA, and HQSAR models have values of r (2) = 0.988, q (2) = 0.757, ONC = 6; r (2) = 0.966, q (2) = 0.645, ONC = 5; and r (2) = 0.957, q (2) = 0.736, ONC = 6. The predictabilities were validated using an external test sets, and the predictive r (2) values obtained by the three models were 0.984, 0.973, and 0.783, respectively. The analysis was performed by combining the CoMFA and CoMSIA field distributions with the active sites of the AChE to further understand the vital interactions between huprines and the protease. On the basis of the QSAR study, 14 new potent molecules have been designed and six of them are predicted to be more active than the best active compound 24 described in the literature. The final QSAR models could be helpful in design and development of novel active AChE inhibitors.

Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation.

PubMed

Kaplan, Mehmet Mahsum; Sultana, Nasreen; Benedetti, Ariane; Obermair, Gerald J; Linde, Nina F; Papadopoulos, Symeon; Dayal, Anamika; Grabner, Manfred; Flucher, Bernhard E

2018-06-26

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (Ca V 1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase.

PubMed

Boll, Matthias; Einsle, Oliver; Ermler, Ulrich; Kroneck, Peter M H; Ullmann, G Matthias

2016-01-01

In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis-pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg2+ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry. © 2016 S. Karger AG, Basel.

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay.

PubMed

Geen, J; Hullin, D A; Hogg, S I

1999-01-01

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.

Comparative functional expression of nAChR subtypes in rodent DRG neurons.

PubMed

Smith, Nathan J; Hone, Arik J; Memon, Tosifa; Bossi, Simon; Smith, Thomas E; McIntosh, J Michael; Olivera, Baldomero M; Teichert, Russell W

2013-01-01

We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70-80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15-30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca(2) (+) transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.

Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

PubMed

Azad, Kimi; Banerjee, Manidipa; Johnson, John E

2017-09-29

Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

A positive allosteric modulator of α7 nAChRs augments neuroprotective effects of endogenous nicotinic agonists in cerebral ischaemia

PubMed Central

Kalappa, Bopanna I; Sun, Fen; Johnson, Stephen R; Jin, Kunlin; Uteshev, Victor V

2013-01-01

Background and Purpose Activation of α7 nicotinic acetylcholine receptors (nAChRs) can be neuroprotective. However, endogenous choline and ACh have not been regarded as potent neuroprotective agents because physiological levels of choline/ACh do not produce neuroprotective levels of α7 activation. This limitation may be overcome by the use of type-II positive allosteric modulators (PAMs-II) of α7 nAChRs, such as 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea (PNU-120596). This proof-of-concept study presents a novel neuroprotective paradigm that converts endogenous choline/ACh into potent neuroprotective agents in cerebral ischaemia by inhibiting α7 nAChR desensitization using PNU-120596. Experimental Approach An electrophysiological ex vivo cell injury assay (to quantify the susceptibility of hippocampal neurons to acute injury by complete oxygen and glucose deprivation; COGD) and an in vivo middle cerebral artery occlusion model of ischaemia were used in rats. Key Results Choline (20–200 μM) in the presence, but not absence of 1 μM PNU-120596 significantly delayed anoxic depolarization/injury of hippocampal CA1 pyramidal neurons, but not CA1 stratum radiatum interneurons, subjected to COGD in acute hippocampal slices and these effects were blocked by 20 nM methyllycaconitine, a selective α7 antagonist, thus, activation of α7 nAChRs was required. PNU-120596 alone was ineffective ex vivo. In in vivo experiments, both pre- and post-ischaemia treatments with PNU-120596 (30 mg·kg−1, s.c. and 1 mg·kg−1, i.v., respectively) significantly reduced the cortical/subcortical infarct volume caused by transient focal cerebral ischaemia. PNU-120596 (1 mg·kg−1, i.v., 30 min post-ischaemia) remained neuroprotective in rats subjected to a choline-deficient diet for 14 days prior to experiments. Conclusions and Implications PNU-120596 and possibly other PAMs-II significantly improved neuronal survival in cerebral ischaemia by augmenting

Congo red modulates ACh-induced Ca2+ oscillations in single pancreatic acinar cells of mice

PubMed Central

Huang, Ze-bing; Wang, Hai-yan; Sun, Na-na; Wang, Jing-ke; Zhao, Meng-qin; Shen, Jian-xin; Gao, Ming; Hammer, Ronald P; Fan, Xue-gong; Wu, Jie

2014-01-01

Aim: Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca2+ oscillations in mouse pancreatic acinar cells in vitro. Methods: Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca2+ oscillations were monitored by whole-cell recording of Ca2+-activated Cl− currents and by using confocal Ca2+ imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established. Results: Bath application of ACh (10 nmol/L) induced typical Ca2+ oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 μmol/L) dose-dependently enhanced Ach-induced Ca2+ oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca2+ oscillations induced by ACh (10–30 nmol/L), but did not alter the Ca2+ oscillations induced by ACh (100–10000 nmol/L). Congo red also enhanced the Ca2+ oscillations induced by bath application of IP3 (30 μmol/L). Intracellular application of congo red failed to alter ACh-induced Ca2+ oscillations. Conclusion: Congo red significantly modulates intracellular Ca2+ signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug. PMID:25345744

Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

PubMed

Bi, Xiaodong; Liu, Zhen

2014-12-16

Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

Cholinesterase activity of muscle tissue from freshwater fishes: characterization and sensitivity analysis to the organophosphate methyl-paraoxon.

PubMed

Lopes, Renato Matos; Filho, Moacelio Veranio Silva; de Salles, João Bosco; Bastos, Vera Lúcia Freire Cunha; Bastos, Jayme Cunha

2014-06-01

The biochemical characterization of cholinesterases (ChE) from different teleost species has been a critical step in ensuring the proper use of ChE activity levels as biomarkers in environmental monitoring programs. In the present study, ChE from Oreochromis niloticus, Piaractus mesopotamicus, Leporinus macrocephalus, and Prochilodus lineatus was biochemically characterized by specific substrates and inhibitors. Moreover, muscle tissue ChE sensitivity to the organophosphate pesticide methyl-paraoxon was evaluated by determining the inhibition kinetic constants for its progressive irreversible inhibition by methyl-paraoxon as well as the 50% inhibitory concentration (IC50) for 30 min for each species. The present results indicate that acetylcholinesterase (AChE) must be present in the muscle from P. mesopotamicus, L. macrocephalus, and P. lineatus and that O. niloticus possesses an atypical cholinesterase or AChE and butyrylcholinesterase (BChE). Furthermore, there is a large difference regarding the sensitivity of these enzymes to methyl-paraoxon. The determined IC50 values for 30 min were 70 nM (O. niloticus), 258 nM (P. lineatus), 319 nM (L. macrocephalus), and 1578 nM (P. mesopotamicus). The results of the present study also indicate that the use of efficient methods for extracting these enzymes, their kinetic characterization, and determination of sensitivity differences between AChE and BChE to organophosphate compounds are essential for the determination of accurate ChE activity levels for environmental monitoring programs. © 2014 SETAC.

Inhibitory effects of psychotropic drugs on the acetylcholine receptor-operated potassium current (IK.ACh) in guinea-pig atrial myocytes.

PubMed

Okada, Muneyoshi; Watanabe, Shinya; Matada, Takashi; Asao, Yoko; Hamatani, Ramu; Yamawaki, Hideyuki; Hara, Yukio

2013-01-01

Influences of psychotropic drugs, six antipsychotics and three antidepressants, on acetylcholine receptor-operated potassium current (IK.ACh) were examined by a whole-cell patch clamp method in freshly isolated guinea-pig atrial myocyte. IK.ACh was induced by a superfusion of carbachol (CCh) or by an intracellular application of guanosine 5'-[thio] triphosphate (GTPγS). To elucidate mechanism for anticholinergic action, IC50 ratio, the ratio of IC50 for GTPγS-activated IK.ACh to CCh-induced IK.ACh, was calculated. Antipsychotics and antidepressants inhibited CCh-induced IK.ACh in a concentration-dependent manner. The IC50 values were as follows; chlorpromazine 0.53 μM, clozapine 0.06 μM, fluphenazine 2.69 μM, haloperidol 2.66 μM, sulpiride 42.3 μM, thioridazine 0.07 μM, amitriptyline 0.03 μM, imipramine 0.22 μM and maprotiline 1.81 μM. The drugs, except for sulpiride, inhibited GTPγS-activated IK.ACh with following IC50 values; chlorpromazine 1.71 μM, clozapine 14.9 μM, fluphenazine 3.55 μM, haloperidol 2.73 μM, thioridazine 1.90 μM, amitriptyline 7.55 μM, imipramine 7.09 μM and maprotiline 5.93 μM. The IC50 ratio for fluphenazine and haloperidol was close to unity. The IC50 ratio for chlorpromazine, clozapine, thioridazine, amitriptyline, imipramine and maprotiline was much higher than unity. The present findings suggest that the psychotropics studied suppress IK.ACh. Chlorpromazine, clozapine, thioridazine, amitriptyline, imipramine, maprotiline and sulpiride are preferentially acting on muscarinic receptor. Fluphenazine and haloperidol may act on G protein and/or potassium channel.

Regional acetylcholinesterase activity and its correlation with behavioral performances in 15-month old transgenic mice expressing the human C99 fragment of APP.

PubMed

Dumont, M; Lalonde, R; Ghersi-Egea, J-F; Fukuchi, K; Strazielle, C

2006-09-01

In addition to Abeta plaques and neurofibrillary tangles, Alzheimer's disease (AD) is characterized by increased brain levels of APP C-terminal fragments. In the present investigation, the cholinergic innervation in forebrain regions of transgenic mice (Tg13592) expressing the human betaAPP C99 fragment was compared to that of non-transgenic controls by measuring the activity of the non-specific catabolic enzyme, acetylcholinesterase (AChE). The AchE activity of Tg13592 mice was altered in several regions implicated in the functional loop of regulation between septum and hippocampus, vulnerable in Alzheimer pathology and critically involved in cognitive functions. In particular, AChE activity was upregulated in three basal forebrain regions containing cholinergic cell bodies, prelimbic cortex, anterior subiculum, and paraventricular thalamus, but downregulated in lateral septum and reticular thalamus. The increased activity in medial septum and anterior subiculum was linearly correlated with poor performances in a spatial learning task, possibly due to cell stress mechanisms. Because of some similarities in terms of neurochemistry and behavior, this mouse model may be of use for studying prodromal AD.

In vitro and in silico Studies of Mangiferin from Aphloia theiformis on Key Enzymes Linked to Diabetes Type 2 and Associated Complications.

PubMed

Picot, Marie C N; Zengin, Gokhan; Mollica, Adriano; Stefanucci, Azzurra; Carradori, Simone; Mahomoodally, Mohamad F

2017-01-01

Mangiferin, was identified in the crude methanol extract, ethyl acetate, and n-butanol fractions of Aphloia theiformis (Vahl.) Benn. This study aimed to analyze the plausible binding modes of mangiferin to key enzymes linked to diabetes type 2 (DT2), obesity, hypertension, Alzheimer's disease, and urolithiasis using molecular docking. Crystallographic structures of α-amylase, α-glucosidase, glycogen phosphorylase (GP), pancreatic lipase, cholesterol esterase (CEase), angiotensin-I-converting enzyme (ACE), acetyl cholinesterase (AChE), and urease available on the Protein Databank database were docked to mangiferin using Gold 6.0 software. We showed that mangiferin bound to all enzymes by π-π and hydrogen bonds mostly. Mangiferin was docked to both allosteric and orthosteric sites of α-glucosidase by π-π interactions. However, several hydrogen bonds were observed at the orthosteric position, suggesting a preference for this site. The docking of mangiferin on AChE with the catalytic pocket occupied by paraoxon could be attributed to π-π stacking involving amino acid residues, Trp341 and Trp124. This study provided an insight of the molecular interaction of mangiferin with the studied enzymes and can be considered as a valuable tool for designing new drugs for better management of these diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Inhibitory potential of some Romanian medicinal plants against enzymes linked to neurodegenerative diseases and their antioxidant activity

PubMed Central

Paun, Gabriela; Neagu, Elena; Albu, Camelia; Radu, Gabriel Lucian

2015-01-01

Context: Eryngium planum, Geum urbanum and Cnicus benedictus plants are an endemic botanical from the Romanian used in folk medicine. Objective: The extracts from three Romanian medicinal plants were investigated for their possible neuroprotective potential. Materials and Methods: Within this study, in vitro neuroprotective activity of the extracts of E. planum, G. urbanum, and C. benedictus plants were investigated via inhibition of acetylcholinesterase (AChE) and tyrosinase (TYR). Total content of phenolics, flavonoids, and proanthocyanidins, high-performance liquid chromatography profile of the main phenolic compounds and antioxidant activity were also determined. Results: Among the tested extracts, the best inhibition of AChE (88.76 ± 5.2%) and TYR (88.5 ± 5.2%) was caused by C. benedictus ethanol (EtOH) extract. The G. urbanum extracts exerted remarkable scavenging effect against 2, 2-diphenyl-1-picrylhydrazyl (IC50, 7.8 ± 0.5 μg/mL aqueous extract, and IC50, 1.3 ± 0.1 μg/mL EtOH extract, respectively) and reducing power, whereas the EtOH extract of C. benedictus showed high scavenging activity (IC50, 0.609 ± 0.04 mg/mL), also. Conclusion: According to our knowledge, this is the first study that demonstrates in vitro neuroprotective effects of E. planum, G. urbanum and C. benedictus. PMID:26109755

EQCM Immunoassay for Phosphorylated Acetylcholinesterase as a Biomarker for Organophosphate Exposures Based on Selective Zirconia Adsorption and Enzyme-Catalytic Precipitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hua; Wang, Jun; Choi, Daiwon

2009-03-01

A zirconia (ZrO2) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO2 film with uniform nanostructures. The resulting ZrO2 film was utilized to selectively capture phosphorylated AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated protein. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulationmore » of insoluble product on the functionalized crystal. Ultrasensitive EQCM quantification by mass-amplified frequency responses as well as rapid qualification by visual color changes of product could be thus achieved. Moreover, 4-chloro-1-naphthol (CN) was comparably studied as an ideal chromogenic substrate for the enzyme-catalytic precipitation. Experimental results show that the developed EQCM technique can allow for the detection of phosphorylated AChE in human plasma. Such an EQCM immunosensing format opens a new door towards the development of simple, sensitive, and field-applicable biosensor for biologically monitoring low-level OP exposures.« less

Determining Enzyme Activity by Radial Diffusion

ERIC Educational Resources Information Center

Davis, Bill D.

1977-01-01

Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

Induction of plasma acetylcholinesterase activity in mice challenged with organophosphorus poisons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duysen, Ellen G.; Lockridge, Oksana, E-mail: olockrid@unmc.edu

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 thanmore » 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.« less

Generation of Recombinant Human AChE OP-Scavengers with Extended Circulatory Longevity

DTIC Science & Technology

2006-11-01

PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Avigdor Shafferman Ph.D. 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail: avigdor...glycans of rHuAChE, and in particular the distal termini of these glycan 8 projections , constitute a major factor in determining the circulatory...experiments funded by sources other than the USAMRMC contract, since they may have a major impact on the present project . Section III focuses on the

Pyridine sulfonamide as a small key organic molecule for the potential treatment of type-II diabetes mellitus and Alzheimer's disease: In vitro studies against yeast α-glucosidase, acetylcholinesterase and butyrylcholinesterase.

PubMed

Riaz, Sadaf; Khan, Islam Ullah; Bajda, Marek; Ashraf, Muhammad; Qurat-Ul-Ain; Shaukat, Ayesha; Rehman, Tanzeel Ur; Mutahir, Sadaf; Hussain, Sajjad; Mustafa, Ghulam; Yar, Muhammad

2015-12-01

This paper presents the efficient high yield synthesis of novel pyridine 2,4,6-tricarbohydrazide derivatives (4a-4i) along with their α-glucosidase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition activities. The enzymes inhibition results showed the potential of synthesized compounds in controlling both type-II diabetes mellitus and Alzheimer's disease. In vitro biological investigations revealed that most of compounds were more active against yeast α-glucosidase than the reference compound acarbose (IC50 38.25±0.12μM). Among the tested series the compound 4c bearing 4-flouro benzyl group was noted to be the most active (IC50 25.6±0.2μM) against α-glucosidase, and it displayed weak inhibition activities against AChE and BChE. Compound 4a exhibited the most desired results against all three enzymes, as it was significantly active against all the three enzymes; α-glucosidase (IC50 32.2±0.3μM), AChE (IC50 50.2±0.8μM) and BChE (IC50 43.8±0.8μM). Due to the most favorable activity of 4a against the tested enzymes, for molecular modeling studies this compound was selected to investigate its pattern of interaction with α-glucosidase and AChE targets. Copyright © 2015 Elsevier Inc. All rights reserved.

Modulatory effects of α7 nAChRs on the immune system and its relevance for CNS disorders.

PubMed

Kalkman, Hans O; Feuerbach, Dominik

2016-07-01

The clinical development of selective alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists has hitherto been focused on disorders characterized by cognitive deficits (e.g., Alzheimer's disease, schizophrenia). However, α7 nAChRs are also widely expressed by cells of the immune system and by cells with a secondary role in pathogen defense. Activation of α7 nAChRs leads to an anti-inflammatory effect. Since sterile inflammation is a frequently observed phenomenon in both psychiatric disorders (e.g., schizophrenia, melancholic and bipolar depression) and neurological disorders (e.g., Alzheimer's disease, Parkinson's disease, and multiple sclerosis), α7 nAChR agonists might show beneficial effects in these central nervous system disorders. In the current review, we summarize information on receptor expression, the intracellular signaling pathways they modulate and reasons for receptor dysfunction. Information from tobacco smoking, vagus nerve stimulation, and cholinesterase inhibition is used to evaluate the therapeutic potential of selective α7 nAChR agonists in these inflammation-related disorders.

Recent advances in acetylcholinesterase Inhibitors and Reactivators: an update on the patent literature (2012-2015).

PubMed

McHardy, Stanton F; Wang, Hua-Yu Leo; McCowen, Shelby V; Valdez, Matthew C

2017-04-01

Acetylcholinesterase (AChE) is the major enzyme that hydrolyzes acetylcholine, a key neurotransmitter for synaptic transmission, into acetic acid and choline. Mild inhibition of AChE has been shown to have therapeutic relevance in Alzheimer's disease (AD), myasthenia gravis, and glaucoma among others. In contrast, strong inhibition of AChE can lead to cholinergic poisoning. To combat this, AChE reactivators have to be developed to remove the offending AChE inhibitor, restoring acetylcholine levels to normal. Areas covered: This article covers recent advances in the development of acetylcholinesterase modulators, including both inhibitors of acetylcholinesterase for the efforts in development of new chemical entities for treatment of AD, as well as re-activators for resurrection of organophosphate bound acetylcholinesterase. Expert opinion: Over the past three years, research efforts have continued to identify novel small molecules as AChE inhibitors for both CNS and peripheral diseases. The more recent patent activity has focused on three AChE ligand design areas: derivatives of known AChE ligands, natural product based scaffolds and multifunctional ligands, all of which have produced some unique chemical matter with AChE inhibition activities in the mid picomolar to low micromolar ranges. New AChE inhibitors with polypharmacology or dual inhibitory activity have also emerged as highlighted by new AChE inhibitors with dual activity at L-type calcium channels, GSK-3, BACE1 and H3, although most only show low micromolar activity, thus further research is warranted. New small molecule reactivators of organophosphate-inhibited AChE have also been disclosed, which focused on the design of neutral ligands with improved pharmaceutical properties and blood-brain barrier (BBB) penetration. Gratifyingly, some research in this area is moving away from the traditional quaternary pyridinium oximes AChE reactivators, while still employing the necessary reactivation group (oximes

Evidence for a role for α6* nAChRs in L-dopa-induced dyskinesias using parkinsonian α6* nAChR gain-of-function mice

PubMed Central

Bordia, Tanuja; McGregor, Matthew; McIntosh, J.M.; Drenan, Ryan M.; Quik, Maryka

2015-01-01

L-Dopa-induced dyskinesias (LIDs) are a serious side effect of dopamine replacement therapy for Parkinson's disease. The mechanisms that underlie LIDs are currently unclear. However, preclinical studies indicate that nicotinic acetylcholine receptors (nAChRs) play a role, suggesting that drugs targeting these receptors may be of therapeutic benefit. To further understand the involvement of α6β2* nAChRs in LIDs, we used gain-of-function α6* nAChR (α6L9S) mice that exhibit a 20-fold enhanced sensitivity to nAChR agonists. Wildtype (WT) and α6L9S mice were lesioned by unilateral injection of 6-hydroxydopamine (6-OHDA, 3 μg/ml) into the medial forebrain bundle. Three to 4 wk later, they were administered L-dopa (3 mg/kg) plus benserazide (15 mg/kg) until stably dyskinetic. L-dopa-induced abnormal involuntary movements (AIMs) were similar in α6L9S and WT mice. WT mice were then given nicotine in the drinking water in gradually increasing doses to a final 300 μg/ml, which resulted in a 40% decline AIMs. By contrast, there was no decrease in AIMs in α6L9S mice at a maximally tolerated nicotine dose of 20 μg/ml. However, the nAChR antagonist mecamylamine (1 mg/kg ip 30 min before L-dopa) reduced L-dopa-induced AIMs in both α6L9S and WT mice. Thus, both a nAChR agonist and antagonist decreased AIMs in WT mice, but only the antagonist was effective in α6L9S mice. Since nicotine appears to reduce LIDs via desensitization, hypersensitive α6β2* nAChRs may desensitize less readily. The present data show that α6β2* nAChRs are key regulators of LIDs, and may be useful therapeutic targets for their management in Parkinson's disease. PMID:25813704

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

PubMed

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

2015-01-01

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

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

PubMed Central

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

2015-01-01

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

Chemical composition, antioxidant properties and anti-cholinesterase activity of Cordia gilletii (Boraginaceae) leaves essential oil.

PubMed

Bonesi, Marco; Okusa, Philippe N; Tundis, Rosa; Loizzo, Monica R; Menichini, Federica; Stévigny, Caroline; Duez, Pierre; Menichini, Francesco

2011-02-01

This study aimed to investigate for the first time the chemical composition, the antioxidant properties and the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity of the essential oil from the leaves of Cordia gilletii De Wild (Boraginaceae). The essential oil, characterized by 23 constituents (90.1% of the total oil), was constituted by terpene derivatives (25.6%) and non-terpene derivatives (64.5%), among which aldehydes, fatty acids and alkanes were present with the percentage of 16.5%, 18.8% and 23.1%, respectively. The antioxidant activity of C. gilletii essential oil was screened by two in vitro tests: DPPH and beta-carotene bleaching test. The essential oil revealed antioxidant activity with an IC50 value of 75.0 and 129.9 microg/mL on DPPH radical and beta-carotene decoloration tests, respectively. Moreover, C. gilletii inhibited AChE enzyme with an IC50 value of 105.6 microg/mL.

Synthesis and biological evaluation of phloroglucinol derivatives possessing α-glycosidase, acetylcholinesterase, butyrylcholinesterase, carbonic anhydrase inhibitory activity.

PubMed

Burmaoglu, Serdar; Yilmaz, Ali O; Taslimi, Parham; Algul, Oztekin; Kilic, Deryanur; Gulcin, Ilhami

2018-02-01

A series of novel phloroglucinol derivatives were designed, synthesized, characterized spectroscopically and tested for their inhibitory activity against selected metabolic enzymes, including α-glycosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human carbonic anhydrase I and II (hCA I and II). These compounds displayed nanomolar inhibition levels and showed K i values of 1.14-3.92 nM against AChE, 0.24-1.64 nM against BChE, 6.73-51.10 nM against α-glycosidase, 1.80-5.10 nM against hCA I, and 1.14-5.45 nM against hCA II. © 2018 Deutsche Pharmazeutische Gesellschaft.

New Indole Alkaloids from the Bark of Rauvolfia Reflexa and their Cholinesterase Inhibitory Activity.

PubMed

Fadaeinasab, Mehran; Basiri, Alireza; Kia, Yalda; Karimian, Hamed; Ali, Hapipah Mohd; Murugaiyah, Vikneswaran

2015-01-01

Rauvolfia reflexa is a member of the Apocynaceae family. Plants from the Apocynaceae family have been traditionally used in the treatment of age-related brain disorders Methods and Results: Two new indole alkaloids, rauvolfine C (1) and 3-methyl-10,11-dimethoxy-6-methoxycarbonyl-β-carboline (2), along with five known, macusine B (3), vinorine (4), undulifoline (5), isoresrpiline (6) and rescinnamine (7) were isolated from the bark of Rauvolfia reflexa. Cholinesterase inhibitory assay and molecular docking were performed to get insight of the inhibitory activity and molecular interactions of the compounds. The compounds showed good to moderate cholinesterase inhibitory activity with IC50 values in the range of 8.06 to 73.23 µM. Compound 7 was found to be the most potent inhibitor of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Compounds 1, 2, 5 and 6 were found to be selective towards BChE, while compounds 3, 4 and 7 were dual inhibitors, having almost equal inhibitory activity on both AChE and BChE. Molecular docking revealed that compounds 6 and 7 interacted differently on AChE and BChE, by means of hydrophobic interactions and hydrogen bonding. In AChE, the indole moiety of both compounds interacted with the residues lining the peripheral anionic site, whereas in BChE, their methoxy groups are primarily responsible for the strong inhibitory activity via interactions with residues at the active site of the enzyme. Two new and five known indole alkaloids were isolated from R. reflexa. Among the compounds, 7 and 6 showed the most potent and promising cholinesterase inhibitory activity, worthy for further investigations. © 2015 S. Karger AG, Basel.

Glycyl radical activating enzymes: Structure, mechanism, and substrate interactions☆

PubMed Central

Shisler, Krista A.; Broderick, Joan B.

2014-01-01

The glycyl radical enzyme activating enzymes (GRE–AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe–4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE–AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE–AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE–AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes. PMID:24486374

9-Substituted acridine derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors possessing antioxidant activity for Alzheimer's disease treatment.

PubMed

Makhaeva, Galina F; Lushchekina, Sofya V; Boltneva, Natalia P; Serebryakova, Olga G; Rudakova, Elena V; Ustyugov, Alexey A; Bachurin, Sergey O; Shchepochkin, Alexander V; Chupakhin, Oleg N; Charushin, Valery N; Richardson, Rudy J

2017-11-01

We investigated the inhibitory activity of 4 groups of novel acridine derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) using the methods of enzyme kinetics and molecular docking. Antioxidant activity of the compounds was determined using the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) radical decolorization assay as their ability to scavenge free radicals. Analysis of the esterase profiles and antiradical activities of the acridine derivatives showed that 9-aryl(heteroaryl)-N-methyl-9,10-dihydroacridines have a high radical-scavenging activity but low potency as AChE and BChE inhibitors, whereas 9-aryl(heteroaryl)-N-methyl-acridinium tetrafluoroborates effectively inhibit cholinesterases but do not exhibit antiradical activity. In contrast, a group of derivatives of 9-heterocyclic amino-N-methyl-9,10-dihydroacridine has been found that combine effective inhibition of AChE and BChE with rather high radical-scavenging activity. The results of molecular docking well explain the observed features in the efficacy, selectivity, and mechanism of cholinesterase inhibition by the acridine derivatives. Thus, in a series of acridine derivatives we have found compounds possessing dual properties of effective and selective cholinesterase inhibition together with free radical scavenging, which makes promising the use of the acridine scaffold to create multifunctional drugs for the therapy of neurodegenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine.

PubMed

Ohta, Kazumasa; Takahashi, Chifumi; Tosuji, Hiroaki

2009-08-01

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.

Enzyme activity in dialkyl phosphate ionic liquids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomas, M.F.; Dunn, J.; Li, L.-L.

2011-12-01

The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

PubMed

Shisler, Krista A; Broderick, Joan B

2014-03-15

The glycyl radical enzyme activating enzymes (GRE-AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe-4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE-AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE-AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE-AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes. Copyright © 2014. Published by Elsevier Inc.

α7-nAChR Knockout Mice Decreases Biliary Hyperplasia and Liver Fibrosis in Cholestatic Bile-Duct Ligated Mice.

PubMed

Ehrlich, Laurent; O'Brien, April; Hall, Chad; White, Tori; Chen, Lixian; Wu, Nan; Venter, Julie; Scrushy, Marinda; Mubarak, Muhammad; Meng, Fanyin; Dostal, David; Wu, Chaodong; Lairmore, Terry C; Alpini, Gianfranco; Glaser, Shannon

2018-03-26

α7-nAChR is a nicotinic acetylcholine receptor (specifically expressed on hepatic stellate cells, Kupffer cells, and cholangiocytes) that regulates inflammation and apoptosis in the liver. Thus, targeting α7-nAChR may be therapeutic in biliary diseases. Bile-duct ligation (BDL) was performed on wild-type (WT) and α7-nAChR-/- mice. We first evaluated the expression of α7-nAChR by immunohistochemistry (IHC) in liver sections. IHC was also performed to assess intrahepatic bile-duct mass (IBDM), and Sirius Red staining was performed to quantify the amount of collagen deposition. Immunofluorescence was performed to assess co-localization of α7-nAChR with bile ducts (co-stained with CK-19) and hepatic stellate cells (HSCs) (co-stained with desmin). The mRNA expression of α7-nAChR, Ki67/PCNA (proliferation), fibrosis genes (TGF-β1, Fibronectin-1, Col1α1, and α-SMA), and inflammatory markers (IL-6, IL-1β, and TNFα) was measured by real-time PCR. Biliary TGF-β1 and hepatic CD68 (Kupffer cell marker) expression was assessed using IHC. α7-nAChR immunoreactivity was observed in both bile ducts and HSCs and increased following BDL. α7-nAChR-/- BDL mice exhibited decreased: (i) bile duct mass, liver fibrosis, and inflammation; and (ii) immunoreactivity of TGF-1 as well as expression of fibrosis genes compared to WT BDL mice. α7-nAChR activation triggers biliary proliferation and liver fibrosis and may be a therapeutic target in managing extra-hepatic biliary obstruction.

Ultastructural analysis on acetylcholinesterase localization in the cerebellar cortex of teleosts.

PubMed

Contestabile, A; Villani, L; Ciani, F

1977-12-28

The histochemical localization of acetylcholinesterase (AChE) was studied by electron microscopy in the cerebellar cortex of the goldfish and the catfish. The patterns of enzyme distribution show noticeable differences in the two teleost species at the level of the corresponding cerebellar structures. Among the most distinctive features is the prevailing intracellular localization of enzyme activity in the goldfish and the prevailing extracellular localization in the catfish in the molecular layer and, to a lesser extent, the granular layer. Only quantitative differences in the ability to synthesize AChE can be recorded among the different cerebellar neurons in the two species, since all these neurons exhibit different amounts of enzyme activity linked to their cytoplasmic structures. Comparing the results obtained with those of previous histochemical, experimental and developmental researches, the hypothesis seems well founded that the embryonic pool of cerebellar neurons is made up of AChE-synthesizing nruroblasts which, during development, loss or maintain to a different the mechanisms for AChE synthesis. In addition the light and electron microscope histochemistry reveals at different levels of resolution that the final pattern of AChE distribution in the cerebellar cortex is the sum of different degress of AChE synthesis by cerebellar neurons and different degrees of enzyme release in extracellular spaces.

Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice

PubMed Central

Cole, R N; Ghazanfari, N; Ngo, S T; Gervásio, O L; Reddel, S W; Phillips, W D

2010-01-01

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day−1i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51–73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR β-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering. PMID:20603331

Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

PubMed

Vulfius, Catherine A; Kasheverov, Igor E; Kryukova, Elena V; Spirova, Ekaterina N; Shelukhina, Irina V; Starkov, Vladislav G; Andreeva, Tatyana V; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I; Utkin, Yuri N

2017-01-01

Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

PubMed

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

PubMed

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

2011-02-17

Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

Nicotine promotes cell proliferation via {alpha}7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee

Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a {beta}{sub 1}- and {beta}{sub 2}-selective antagonist, respectively, suggesting the role of {beta}-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-{beta}-hydroxylase (D{beta}H) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferationmore » and adrenaline production. Expression of {alpha}7-nicotinic acetylcholine receptor ({alpha}7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an {alpha}7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and D{beta}H expression as well as adrenaline production. Taken together, through the action on {alpha}7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and {beta}-adrenergic activation. These data reveal the contributory role {alpha}7-nAChR and {beta}-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer.« less

A Disposable Organophosphorus Pesticides Enzyme Biosensor Based on Magnetic Composite Nano-Particles Modified Screen Printed Carbon Electrode

PubMed Central

Gan, Ning; Yang, Xin; Xie, Donghua; Wu, Yuanzhao; Wen, Weigang

2010-01-01

A disposable organophosphorus pesticides (OPs) enzyme biosensor based on magnetic composite nanoparticle-modified screen printed carbon electrodes (SPCE) has been developed. Firstly, an acetylcholinesterase (AChE)-coated Fe3O4/Au (GMP) magnetic nanoparticulate (GMP-AChE) was synthesized. Then, GMP-AChE was absorbed on the surface of a SPCE modified by carbon nanotubes (CNTs)/nano-ZrO2/prussian blue (PB)/Nafion (Nf) composite membrane by an external magnetic field. Thus, the biosensor (SPCE│CNTs/ZrO2/PB/Nf│GMP-AChE) for OPs was fabricated. The surface of the biosensor was characterized by scanning electron micrography (SEM) and X-ray fluorescence spectrometery (XRFS) and its electrochemical properties were studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The degree of inhibition (A%) of the AChE by OPs was determined by measuring the reduction current of the PB generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh). In pH = 7.5 KNO3 solution, the A was related linearly to the concentration of dimethoate in the range from 1.0 × 10−3–10 ng·mL−1 with a detection limit of 5.6 × 10−4 ng·mL−1. The recovery rates in Chinese cabbage exhibited a range of 88%–105%. The results were consistent with the standard gas chromatography (GC) method. Compared with other enzyme biosensors the proposed biosensor exhibited high sensitivity, good selectivity with disposable, low consumption of sample. In particular its surface can be easily renewed by removal of the magnet. The convenient, fast and sensitive voltammetric measurement opens new opportunities for OPs analysis. PMID:22315558

Anthocyanins as a potential pharmacological agent to manage memory deficit, oxidative stress and alterations in ion pump activity induced by experimental sporadic dementia of Alzheimer's type.

PubMed

Pacheco, Simone Muniz; Soares, Mayara Sandrielly Pereira; Gutierres, Jessié Martins; Gerzson, Mariana Freire Barbieri; Carvalho, Fabiano Barbosa; Azambuja, Juliana Hofstatter; Schetinger, Maria Rosa Chitolina; Stefanello, Francieli Moro; Spanevello, Roselia Maria

2018-06-01

Anthocyanins (ANT) are polyphenolic flavonoids with antioxidant and neuroprotective properties. This study evaluated the effect of ANT treatment on cognitive performance and neurochemical parameters in an experimental model of sporadic dementia of Alzheimer's type (SDAT). Adult male rats were divided into four groups: control (1 ml/kg saline, once daily, by gavage), ANT (200 mg/kg, once daily, by gavage), streptozotocin (STZ, 3 mg/kg) and STZ plus ANT. STZ was administered via bilateral intracerebroventricular (ICV) injection (5 μl). ANT were administered after ICV injection for 25 days. Cognitive deficits (short-term memory and spatial memory), oxidative stress parameters, and acetylcholinesterase (AChE) and Na + -K + -ATPase activity in the cerebral cortex and hippocampus were evaluated. ANT treatment protected against the worsening of memory in STZ-induced SDAT. STZ promoted an increase in AChE and Na + -K + -ATPase total and isoform activity in both structures; ANT restored this change. STZ administration induced an increase in lipid peroxidation and decrease in the level of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), in the cerebral cortex; ANT significantly attenuated these effects. In the hippocampus, an increase in reactive oxygen species (ROS), nitrite and lipid peroxidation levels, and SOD activity and a decrease in CAT and GPx activity were seen after STZ injection. ANT protected against the changes in ROS and antioxidant enzyme levels. In conclusion, the present study showed that treatment with ANT attenuated memory deficits, protected against oxidative damage in the brain, and restored AChE and ion pump activity in an STZ-induced SDAT in rats. Copyright © 2018 Elsevier Inc. All rights reserved.

An improved immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand screening.

PubMed

Vilela, Adriana Ferreira Lopes; Seidl, Cláudia; Lima, Juliana Maria; Cardoso, Carmen Lúcia

2018-05-15

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H] + m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC 50 values were 0.03 ± 0.006 μM and 0.88 ± 0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ± 0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results. Copyright © 2018. Published by Elsevier Inc.

Assessment of the expression and role of the α1-nAChR subunit in efferent cholinergic function during the development of the mammalian cochlea.

PubMed

Roux, Isabelle; Wu, Jingjing Sherry; McIntosh, J Michael; Glowatzki, Elisabeth

2016-08-01

Hair cell (HC) activity in the mammalian cochlea is modulated by cholinergic efferent inputs from the brainstem. These inhibitory inputs are mediated by calcium-permeable nicotinic acetylcholine receptors (nAChRs) containing α9- and α10-subunits and by subsequent activation of calcium-dependent potassium channels. Intriguingly, mRNAs of α1- and γ-nAChRs, subunits of the "muscle-type" nAChR have also been found in developing HCs (Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. J Neurosci 35: 5870-5883, 2015; Scheffer D, Sage C, Plazas PV, Huang M, Wedemeyer C, Zhang DS, Chen ZY, Elgoyhen AB, Corey DP, Pingault V. J Neurochem 103: 2651-2664, 2007; Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Sci Rep 1: 26, 2011) prompting proposals that another type of nAChR is present and may be critical during early synaptic development. Mouse genetics, histochemistry, pharmacology, and whole cell recording approaches were combined to test the role of α1-nAChR subunit in HC efferent synapse formation and cholinergic function. The onset of α1-mRNA expression in mouse HCs was found to coincide with the onset of the ACh response and efferent synaptic function. However, in mouse inner hair cells (IHCs) no response to the muscle-type nAChR agonists (±)-anatoxin A, (±)-epibatidine, (-)-nicotine, or 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was detected, arguing against the presence of an independent functional α1-containing muscle-type nAChR in IHCs. In α1-deficient mice, no obvious change of IHC efferent innervation was detected at embryonic day 18, contrary to the hyperinnervation observed at the neuromuscular junction. Additionally, ACh response and efferent synaptic activity were detectable in α1-deficient IHCs, suggesting that α1 is not necessary for assembly and membrane targeting of nAChRs or for efferent synapse formation in IHCs.

Assessment of the expression and role of the α1-nAChR subunit in efferent cholinergic function during the development of the mammalian cochlea

PubMed Central

Wu (武靜靜), Jingjing Sherry; McIntosh, J. Michael; Glowatzki, Elisabeth

2016-01-01

Hair cell (HC) activity in the mammalian cochlea is modulated by cholinergic efferent inputs from the brainstem. These inhibitory inputs are mediated by calcium-permeable nicotinic acetylcholine receptors (nAChRs) containing α9- and α10-subunits and by subsequent activation of calcium-dependent potassium channels. Intriguingly, mRNAs of α1- and γ-nAChRs, subunits of the “muscle-type” nAChR have also been found in developing HCs (Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. J Neurosci 35: 5870–5883, 2015; Scheffer D, Sage C, Plazas PV, Huang M, Wedemeyer C, Zhang DS, Chen ZY, Elgoyhen AB, Corey DP, Pingault V. J Neurochem 103: 2651–2664, 2007; Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Sci Rep 1: 26, 2011) prompting proposals that another type of nAChR is present and may be critical during early synaptic development. Mouse genetics, histochemistry, pharmacology, and whole cell recording approaches were combined to test the role of α1-nAChR subunit in HC efferent synapse formation and cholinergic function. The onset of α1-mRNA expression in mouse HCs was found to coincide with the onset of the ACh response and efferent synaptic function. However, in mouse inner hair cells (IHCs) no response to the muscle-type nAChR agonists (±)-anatoxin A, (±)-epibatidine, (−)-nicotine, or 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was detected, arguing against the presence of an independent functional α1-containing muscle-type nAChR in IHCs. In α1-deficient mice, no obvious change of IHC efferent innervation was detected at embryonic day 18, contrary to the hyperinnervation observed at the neuromuscular junction. Additionally, ACh response and efferent synaptic activity were detectable in α1-deficient IHCs, suggesting that α1 is not necessary for assembly and membrane targeting of nAChRs or for efferent synapse formation in IHCs. PMID:27098031

Combined in Vitro and in Silico Studies for the Anticholinesterase Activity and Pharmacokinetics of Coumarinyl Thiazoles and Oxadiazoles

PubMed Central

Ibrar, Aliya; Khan, Ajmal; Ali, Majid; Sarwar, Rizwana; Mehsud, Saifullah; Farooq, Umar; Halimi, Syed M. A.; Khan, Imtiaz; Al-Harrasi, Ahmed

2018-01-01

In a continuation of our previous work for the exploration of novel enzyme inhibitors, two new coumarin-thiazole 6(a–o) and coumarin-oxadiazole 11(a–h) hybrids have been designed and synthesized. All the compounds were characterized by 1H- and 13C-NMR spectroscopy and elemental analysis. New hybrid analogs were evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in order to know their potential for the prevention of Alzheimer's disease (AD). In coumarinyl thiazole series, compound 6b was found as the most active member against AChE having IC50 value of 0.87 ± 0.09 μM, while the compound 6j revealed the same efficacy against BuChE with an IC50 value of 11.01 ± 3.37 μM. In case of coumarinyl oxadiazole series, 11a was turned out to be the lead candidate against AChE with an IC50 value of 6.07 ± 0.23 μM, whereas compound 11e was found significantly active against BuChE with an IC50 value of 0.15 ± 0.09 μM. To realize the binding interaction of these compounds with AChE and BuChE, the molecular docking studies were performed. Compounds from coumarinyl thiazole series with potent AChE activity (6b, 6h, 6i, and 6k) were found to interact with AChE in the active site with MOE score of −10.19, −9.97, −9.68, and −11.03 Kcal.mol−1, respectively. The major interactions include hydrogen bonding, π-π stacking with aromatic residues, and interaction through water bridging. The docking studies of coumarinyl oxadiazole derivatives 11(a–h) suggested that the compounds with high anti-butyrylcholinesterase activity (11e, 11a, and 11b) provided MOE score of −9.9, −7.4, and −8.2 Kcal.mol−1, respectively, with the active site of BuChE building π-π stacking with Trp82 and water bridged interaction. PMID:29632858

Combined in vitro and in silico studies for the anticholinesterase activity and pharmacokinetics of coumarinyl thiazoles and oxadiazoles

NASA Astrophysics Data System (ADS)

Ibrar, Aliya; Khan, Ajmal; Ali, Majid; Sarwar, Rizwana; Mehsud, Saifullah; Farooq, Umar; Halimi, Syed M. A.; Khan, Imtiaz; Al-Harrasi, Ahmed

2018-03-01

In a continuation of our previous work for the exploration of novel enzyme inhibitors, two new coumarin-thiazole 6(a–o) and coumarin-oxadiazole 11(a–h) hybrids have been designed and synthesized. All the compounds were characterized by 1H- and 13C-NMR spectroscopy and elemental analysis. New hybrid analogues were evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in order to know their potential for the prevention of Alzheimer’s disease (AD). In coumarinyl thiazole series, compound 6b was found as the most active member against AChE having IC50 value of 0.87 ± 0.09 µM, while the compound 6j revealed the same efficacy against BuChE with an IC50 value of 11.01 ± 3.37 µM. In case of coumarinyl oxadiazole series, 11a was turned out to be the lead candidate against AChE with an IC50 value of 6.07 ± 0.23 µM, whereas compound 11e was found significantly active against BuChE with an IC50 value of 0.15 ± 0.09 µM. To realize the binding interaction of these compounds with AChE and BuChE, the molecular docking studies were performed. Compounds from coumarinyl thiazole series with potent AChE activity (6b, 6h, 6i and 6k) were found to interact with AChE in the active site with MOE score of ‒10.19, ‒9.97, ‒9.68, and ‒11.03 Kcal.mol‒1, respectively. The major interactions include hydrogen bonding, π-π stacking with aromatic residues, and interaction through water bridging. The docking studies of coumarinyl oxadiazole derivatives 11(a-h) suggested that the compounds with high anti-butyrylcholinesterase activity (11e, 11a and 11b) provided MOE score of ‒9.9, ‒7.4 and ‒8.2 Kcal.mol‒1 respectively, with the active site of BuChE building π-π stacking with Trp82 and water bridged interaction.

Antioxidant Activity, Acetylcholinesterase, and Carbonic Anhydrase Inhibitory Properties of Novel Ureas Derived from Phenethylamines.

PubMed

Aksu, Kadir; Özgeriş, Bünyamin; Taslimi, Parham; Naderi, Ali; Gülçin, İlhami; Göksu, Süleyman

2016-12-01

A series of ureas derived from phenethylamines were synthesized and evaluated for human carbonic anhydrase (hCA) I and II, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzyme inhibitory activities and antioxidant properties. The ureas were synthesized from the reactions of substituted phenethylamines with N,N-dimethylcarbamoyl chloride; then, the synthesized compounds were converted to their corresponding phenolic derivatives via O-demethylation. hCA I and II were effectively inhibited by the newly synthesized compounds, with K i values in the range of 0.307-0.432 nM for hCA I and 0.149-0.278 nM for hCA II. On the other hand, the K i parameters of these compounds for AChE and BChE were determined in the range of 0.129-0.434 and 0.095-0.207 nM, respectively. Phenolic ureas also showed good antioxidant activities. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of the α7 nicotinic ACh receptor induces anxiogenic effects in rats which is blocked by a 5-HT1a receptor antagonist

PubMed Central

Pandya, Anshul A.; Yakel, Jerrel L.

2013-01-01

The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in different regions of the brain and is associated with cognitive function as well as anxiety. Agonists and positive allosteric modulators (PAMs) of the α7 subtype of nAChRs have been shown to improve cognition. Previously nicotine, which activates both α7 and non-α7 subtypes of nAChRs, has been shown to have an anxiogenic effect in behavioral tests. In this study, we compared the effects of the α7-selective agonist (PNU-282987) and PAM (PNU-120596) in a variety of behavioral tests in Sprague Dawley rats to look at their effects on learning and memory as well as anxiety. We found that neither PNU-282987 nor PNU-120596 improved spatial-learning or episodic memory by themselves. However when cognitive impairment was induced in the rats with scopolamine (1 mg/kg), both PNU-120596 and PNU-282987 were able to reverse this memory impairment and restore it back to normal levels. While PNU-120596 reversed the scopolamine-induced cognitive impairment, it did not have any adverse effect on anxiety. PNU-282987 on the other hand displayed an increase in anxiety-like behavior at a higher dose (10 mg/kg) that was significantly reduced by the serotonin 5-HT1a receptor antagonist WAY-100135. However the α7 receptor antagonist methyllycaconitine was unable to reverse these anxiety-like effects seen with PNU-282987. These results suggest that α7 nAChR PAMs are pharmacologically advantageous over agonists, and should be considered for further development as therapeutic drugs targeting the α7 receptors. PMID:23321689

Integrated Lateral Flow Test Strip with Electrochemical Sensor for Quantification of Phosphorylated Cholinesterase: Biomarker of Exposure to Organophosphorus Agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Dan; Wang, Jun; Wang, Limin

An integrated lateral flow test strip with electrochemical sensor (LFTSES) device with rapid, selective and sensitive response for quantification of exposure to organophosphorus (OP) pesticides and nerve agents has been developed. The principle of this approach is based on parallel measurements of post-exposure and baseline acetylcholinesterase (AChE) enzyme activity, where reactivation of the phosphorylated AChE is exploited to enable measurement of total amount of AChE (including inhibited and active) which is used as a baseline for calculation of AChE inhibition. Quantitative measurement of phosphorylated adduct (OP-AChE) was realized by subtracting the active AChE from the total amount of AChE. Themore » proposed LFTSES device integrates immunochromatographic test strip technology with electrochemical measurement using a disposable screen printed electrode which is located under the test zone. It shows linear response between AChE enzyme activity and enzyme concentration from 0.05 to 10 nM, with detection limit of 0.02 nM. Based on this reactivation approach, the LFTSES device has been successfully applied for in vitro red blood cells inhibition studies using chlorpyrifos oxon as a model OP agent. This approach not only eliminates the difficulty in screening of low-dose OP exposure because of individual variation of normal AChE values, but also avoids the problem in overlapping substrate specificity with cholinesterases and avoids potential interference from other electroactive species in biological samples. It is baseline free and thus provides a rapid, sensitive, selective and inexpensive tool for in-field and point-of-care assessment of exposures to OP pesticides and nerve agents.« less

Enzyme Activity Experiments Using a Simple Spectrophotometer

ERIC Educational Resources Information Center

Hurlbut, Jeffrey A.; And Others

1977-01-01

Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

Compounds from Silicones Alter Enzyme Activity in Curing Barnacle Glue and Model Enzymes

PubMed Central

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H.

2011-01-01

Background Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. Methodology/Principal Findings GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Conclusions/Significance Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management. PMID:21379573

Correlation Among Soil Enzyme Activities, Root Enzyme Activities, and Contaminant Removal in Two-Stage In Situ Constructed Wetlands Purifying Domestic Wastewater.

PubMed

Ni, Lixiao; Xu, Jiajun; Chu, Xianglin; Li, Shiyin; Wang, Peifang; Li, Yiping; Li, Yong; Zhu, Liang; Wang, Chao

2016-07-01

Two-stage in situ wetlands (two vertical flow constructed wetlands in parallel and a horizontal flow constructed wetland) were constructed for studying domestic wastewater purification and the correlations between contaminant removal and plant and soil enzyme activities. Results indicated the removal efficiency of NH4 (+) and NO3 (-) were significantly correlated with both urease and protease activity, and the removal of total phosphorus was significantly correlated with phosphatase activity. Chemical oxygen demand removal was not correlated with enzyme activity in constructed wetlands. Plant root enzyme (urease, phosphatase, protease and cellulose) activity correlation was apparent with all contaminant removal in the two vertical flow constructed wetlands. However, the correlation between the plant root enzyme activity and contaminant removal was poor in horizontal flow constructed wetlands. Results indicated that plant roots clearly played a role in the removal of contaminants.

Activity assessment of microbial fibrinolytic enzymes.

PubMed

Kotb, Essam

2013-08-01

Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

Activation of immobilized enzymes by acoustic wave resonance oscillation.

PubMed

Nishiyama, Hiroshi; Watanabe, Tomoya; Inoue, Yasunobu

2014-12-01

Acoustic wave resonance oscillation has been used successfully in the development of methods to activate immobilized enzyme catalysts. In this study, resonance oscillation effects were demonstrated for enzyme reactions on galactose oxidase (GAD), D-amino acid oxidase (DAAO), and L-amino acid oxidase (LAAO), all of which were immobilized covalently on a ferroelectric lead zirconate titanate (PZT) device that could generate thickness-extensional resonance oscillations (TERO) of acoustic waves. For galactose oxidation on immobilized GAD in a microreactor, TERO generation immediately increased enzyme activity 2- to 3-fold. Eliminating TERO caused a slight decrease in the activity, with ∼90% of the enhanced activity retained while the reaction proceeded. Contact of the enhanced enzyme with a galactose-free solution caused almost complete reversion of the activity to the original low level before TERO generation, indicating that, not only TERO-induced GAD activation, but also preservation of the increased activity, required a galactose substrate. Similar activity changes with TERO were observed for enzyme reactions on DAAO and LAAO. Kinetic analysis demonstrated that TERO helped strengthen the interactions of the immobilized enzyme with the reactant substrate and promoted formation of an activation complex. Copyright © 2014 Elsevier Inc. All rights reserved.

[Women's approach to childhood toothache, abdominal ache and earache].

PubMed

Efe, Emine; Öncel, Selma; Yilmaz, Mualla

2012-01-01

This study was conducted to determine women's about attitudes child's teeth, abdomen and ear ache. Those who had lived in Antalya that 6 number primary health care center between March-May 2004 were enrolled in the study. As data collecting tools. A questionnaire prepared by the authors. This study was determined that 29.2 % of the mothers carried out mixture who had prepared at home to child's abdomen and foot base; 30.3 % were to put breast milk childs' ear; 38.9 % were placed aspirin, salt and salts of lemon to childs' teeth ache. The majority of the women make a wrong practices child that teeth, abdomen and ear ache. This traditional practice effecting factors were the women's educational status and age. The results of the study that education about child care, common health problems and incorrect applications shoud be given to women by nurse.

Normal Modes Expose Active Sites in Enzymes.

PubMed

Glantz-Gashai, Yitav; Meirson, Tomer; Samson, Abraham O

2016-12-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes.

Normal Modes Expose Active Sites in Enzymes

PubMed Central

Glantz-Gashai, Yitav; Samson, Abraham O.

2016-01-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

ORENZA: a web resource for studying ORphan ENZyme activities

PubMed Central

Lespinet, Olivier; Labedan, Bernard

2006-01-01

Background Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. Description We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases. Conclusion ORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities) and sequence (dataset present in public databases). ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme activities by allocating gene

Biphasic photoelectrochemical sensing strategy based on in situ formation of CdS quantum dots for highly sensitive detection of acetylcholinesterase activity and inhibition.

PubMed

Hou, Ting; Zhang, Lianfang; Sun, Xinzhi; Li, Feng

2016-01-15

Herein, we reported a facile and highly sensitive biphasic photoelectrochemical (PEC) sensing strategy based on enzymatic product-mediated in situ formation of CdS quantum dots (QDs), and assayed the activity and inhibition of acetylcholinesterase (AChE) in its optimal state. Upon the hydrolysis of acetylthiocholine catalyzed by AChE, the product thiocholine stabilizes the in situ formation of CdS QDs in homogenous solution. Due to the electrostatic attraction, the resulting tertiary amino group-functionalized CdS QDs are attached to the surface of the negatively charged indium tin oxide (ITO) electrode, generating significant PEC response upon illumination in the presence of electron donors. By taking full advantage of the in situ formation of CdS QDs in homogenous solution, this strategy is capable of detecting AChE activity and inhibition in its optimal state. A directly measured detection limit of 0.01mU/mL for AChE activity is obtained, which is superior to those obtained by some fluorescence methods. The inhibition of AChE activity by aldicarb is successfully detected, and the corresponding IC50 is determined to be 13μg/L. In addition to high sensitivity and good selectivity, this strategy also exhibits additional advantages of simplicity, low cost and easy operation. To the best of our knowledge, the as-proposed strategy is the first example demonstrating the application of CdS QDs formed in situ for biphasic PEC detection of enzyme activity and inhibition. More significantly, it opens up a new horizon for the development of homogenous PEC sensing platforms, and has great potential in probing many other analytes. Copyright © 2015 Elsevier B.V. All rights reserved.

Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in Vitro and in Silico Assays.

PubMed

Liu, Yanyan; Yan, Bing; Winkler, David A; Fu, Jianjie; Zhang, Aiqian

2017-06-07

Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate and product entering and exiting has been recognized previously as an AChE-inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here, we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C 60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C 60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shaped plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modeling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and the curative effect of NPs.

3D MI-DRAGON: new model for the reconstruction of US FDA drug- target network and theoretical-experimental studies of inhibitors of rasagiline derivatives for AChE.

PubMed

Prado-Prado, Francisco; García-Mera, Xerardo; Escobar, Manuel; Alonso, Nerea; Caamaño, Olga; Yañez, Matilde; González-Díaz, Humberto

2012-01-01

The number of neurodegenerative diseases has been increasing in recent years. Many of the drug candidates to be used in the treatment of neurodegenerative diseases present specific 3D structural features. An important protein in this sense is the acetylcholinesterase (AChE), which is the target of many Alzheimer's dementia drugs. Consequently, the prediction of Drug-Protein Interactions (DPIs/nDPIs) between new drug candidates and specific 3D structure and targets is of major importance. To this end, we can use Quantitative Structure-Activity Relationships (QSAR) models to carry out a rational DPIs prediction. Unfortunately, many previous QSAR models developed to predict DPIs take into consideration only 2D structural information and codify the activity against only one target. To solve this problem we can develop some 3D multi-target QSAR (3D mt-QSAR) models. In this study, using the 3D MI-DRAGON technique, we have introduced a new predictor for DPIs based on two different well-known software. We have used the MARCH-INSIDE (MI) and DRAGON software to calculate 3D structural parameters for drugs and targets respectively. Both classes of 3D parameters were used as input to train Artificial Neuronal Network (ANN) algorithms using as benchmark dataset the complex network (CN) made up of all DPIs between US FDA approved drugs and their targets. The entire dataset was downloaded from the DrugBank database. The best 3D mt-QSAR predictor found was an ANN of Multi-Layer Perceptron-type (MLP) with profile MLP 37:37-24-1:1. This MLP classifies correctly 274 out of 321 DPIs (Sensitivity = 85.35%) and 1041 out of 1190 nDPIs (Specificity = 87.48%), corresponding to training Accuracy = 87.03%. We have validated the model with external predicting series with Sensitivity = 84.16% (542/644 DPIs; Specificity = 87.51% (2039/2330 nDPIs) and Accuracy = 86.78%. The new CNs of DPIs reconstructed from US FDA can be used to explore large DPI databases in order to discover both new drugs

Rosuvastatin ameliorates cognitive impairment in rats fed with high-salt and cholesterol diet via inhibiting acetylcholinesterase activity and amyloid beta peptide aggregation.

PubMed

Husain, I; Akhtar, M; Abdin, M Zainul; Islamuddin, M; Shaharyar, M; Najmi, A K

2018-04-01

Amyloid beta (Aβ) peptide aggregation and cholinergic neurodegeneration are involved in the development of cognitive impairment. Therefore, in this article, we examined rosuvastatin (RSV), an oral hypolipidemic drug, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aβ peptide aggregation for the treatment of cognitive impairment. Molecular docking study was done to examine the affinity of RSV with Aβ 1-42 and AChE in silico. We also employed neurobehavioral activity tests, biochemical estimation, and histopathology to study the anti-Aβ 1-42 aggregation capability of RSV in vivo. Molecular docking study provided evidence that RSV has the best binding conformer at its receptor site or active site of an enzyme. The cognitive impairment in female Wistar rats was induced by high-salt and cholesterol diet (HSCD) ad libitum for 8 weeks. RSV ameliorated serum cholesterol level, AChE activity, and Aβ 1-42 peptide aggregations in HSCD induced cognitive impairment. In addition, RSV-treated rats showed greater scores in the open field (locomotor activity) test. Moreover, the histopathological studies in the hippocampus and cortex of rat brain also supported that RSV markedly reduced the cognitive impairment and preserved the normal histoarchitectural pattern of the hippocampus and cortex. Taken together, these data indicate that RSV may act as a dual inhibitor of AChE and Aβ 1-42 peptide aggregation, therefore suggesting a therapeutic strategy for cognitive impairment treatment.

Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody.

PubMed

Morsch, Marco; Reddel, Stephen W; Ghazanfari, Nazanin; Toyka, Klaus V; Phillips, William D

2013-05-15

In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft.

Electrical coupling and release of K+ from endothelial cells co-mediate ACh-induced smooth muscle hyperpolarization in guinea-pig inner ear artery

PubMed Central

Jiang, Zhi-Gen; Nuttall, Alfred L; Zhao, Hui; Dai, Chun-Fu; Guan, Bing-Cai; Si, Jun-Qiang; Yang, Yu-Qin

2005-01-01

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than −60 mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by Nω-nitro-l-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba2+ (100 μm) and ouabain (1 μm) each attenuated ACh-hyperpolarization by ∼ 30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba2+ and 18β-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K+ (10 mm) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49: 1. Ba2+ blocked the K+-induced hyperpolarization by ∼ 85% in both cell types, whereas ouabain inhibited K+-hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18β-Glycyrrhetinic acid blocked the high K+-hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca2+-activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread of the hyperpolarization from the endothelium, and (b

Electrical coupling and release of K+ from endothelial cells co-mediate ACh-induced smooth muscle hyperpolarization in guinea-pig inner ear artery.

PubMed

Jiang, Zhi-Gen; Nuttall, Alfred L; Zhao, Hui; Dai, Chun-Fu; Guan, Bing-Cai; Si, Jun-Qiang; Yang, Yu-Qin

2005-04-15

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than -60 mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by N(omega)-nitro-L-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba(2)(+) (100 microm) and ouabain (1 microm) each attenuated ACh-hyperpolarization by approximately 30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba(2)(+) and 18beta-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K(+) (10 mm) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49 : 1. Ba(2)(+) blocked the K(+)-induced hyperpolarization by approximately 85% in both cell types, whereas ouabain inhibited K(+)-hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18beta-Glycyrrhetinic acid blocked the high K(+)-hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca(2)(+)-activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread

O 2 Activation by Non-Heme Iron Enzymes

DOE PAGES

Solomon, Edward I.; Goudarzi, Serra; Sutherlin, Kyle D.

2016-10-28

The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods have now been developed that provide significant insight into the correlation of structure with function. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III-OOHmore » non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV=O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Lastly, for several subclasses of non-heme Fe enzymes, substrate binding to the Fe II site leads to the one electron reductive activation of O 2 to an Fe III-superoxide capable of H-atom abstraction and electrophilic attack.« less

O 2 Activation by Non-Heme Iron Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solomon, Edward I.; Goudarzi, Serra; Sutherlin, Kyle D.

The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods have now been developed that provide significant insight into the correlation of structure with function. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III-OOHmore » non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV=O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Lastly, for several subclasses of non-heme Fe enzymes, substrate binding to the Fe II site leads to the one electron reductive activation of O 2 to an Fe III-superoxide capable of H-atom abstraction and electrophilic attack.« less

31 CFR 363.38 - What happens if my financial institution returns an ACH debit?

Code of Federal Regulations, 2010 CFR

2010-07-01

... TreasuryDirect § 363.38 What happens if my financial institution returns an ACH debit? If your designated financial institution returns an ACH debit, we reserve the right to reinitiate the debit at our option. We.... We are not responsible for any fees your financial institution may charge relating to returned ACH...

Enhancement of Attentional Performance by Selective Stimulation of α4β2* nAChRs: Underlying Cholinergic Mechanisms

PubMed Central

Howe, William M; Ji, Jinzhao; Parikh, Vinay; Williams, Sarah; Mocaër, Elisabeth; Trocmé-Thibierge, Caryn; Sarter, Martin

2010-01-01

Impairments in attention are a major component of the cognitive symptoms of neuropsychiatric and neurodegenerative disorders. Using an operant sustained attention task (SAT), including a distractor condition (dSAT), we assessed the putative pro-attentional effects of the selective α4β2* nicotinic acetylcholine receptor (nAChR) agonist S 38232 in comparison with the non-selective agonist nicotine. Neither drug benefited SAT performance. However, in interaction with the increased task demands implemented by distractor presentation, the selective agonist, but not nicotine, enhanced the detection of signals during the post-distractor recovery period. This effect is consistent with the hypothesis that second-long increases in cholinergic activity (‘transients') mediate the detection of cues and that nAChR agonists augment such transients. Electrochemical recordings of prefrontal cholinergic transients evoked by S 38232 and nicotine indicated that the α4β2* nAChR agonist evoked cholinergic transients that were characterized by a faster rise time and more rapid decay than those evoked by nicotine. Blockade of the α7 nAChR ‘sharpens' nicotine-evoked transients; therefore, we determined the effects of co-administration of nicotine and the α7 nAChR antagonist methyllycaconitine on dSAT performance. Compared with vehicle and nicotine alone, this combined treatment significantly enhanced the detection of signals. These results indicate that compared with nicotine, α4β2* nAChR agonists significantly enhance attentional performance and that the dSAT represents a useful behavioral screening tool. The combined behavioral and electrochemical evidence supports the hypothesis that nAChR agonist-evoked cholinergic transients, which are characterized by rapid rise time and fast decay, predict robust drug-induced enhancement of attentional performance. PMID:20147893

Synthesis, Biological Evaluation and Molecular Docking Study of Hydrazone-Containing Pyridinium Salts as Cholinesterase Inhibitors.

PubMed

Parlar, Sulunay; Bayraktar, Gulsah; Tarikogullari, Ayse Hande; Alptüzün, Vildan; Erciyas, Ercin

2016-01-01

A series of pyridinium salts bearing alkylphenyl groups at 1 position and hydrazone structure at 4 position of the pyridinium ring were synthesized and evaluated for the inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. The cholinesterase (ChE) inhibitory activity studies were carried out by using the Ellman's colorimetric method. All compounds displayed considerable AChE and BuChE inhibitory activity and some of the compounds manifested remarkable anti-AChE activity compared to the reference compound, galantamine. Among the title compounds, the series including benzofuran aromatic ring exhibited the best inhibitory activity both on AChE and BuChE enzymes. Compound 3b, 4-[2-(1-(benzofuran-2-yl)ethylidene)hydrazinyl]-1-(3-phenylpropyl)pyridinium bromide, was the most active compound with IC50 value of 0.23 (0.24) µM against enantiomeric excess (ee)AChE (human (h)AChE) while compound 3a, 4-[2-(1-(benzofuran-2-yl)ethylidene)hydrazinyl]-1-phenethylpyridinium bromide, was the most active compound with IC50 value of 0.95 µM against BuChE. Moreover, 3a and b exhibited higher activity than the reference compound galantamine (eeAChE (hAChE) IC50 0.43 (0.52) µM; BuChE IC50 14.92 µM). Molecular docking studies were carried out on 3b having highest inhibitory activity against AChE.

Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin

PubMed Central

Ihara, Makoto; Okajima, Toshihide; Yamashita, Atsuko; Oda, Takuma; Hirata, Koichi; Nishiwaki, Hisashi; Morimoto, Takako; Akamatsu, Miki; Ashikawa, Yuji; Kuroda, Shun’ichi; Mega, Ryosuke; Kuramitsu, Seiki; Sattelle, David B.

2008-01-01

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs. PMID:18338186

Cholinesterase activities and behavioral changes in Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed to glufosinate ammonium herbicide.

PubMed

Peltzer, Paola M; Junges, Celina M; Attademo, Andrés M; Bassó, Agustín; Grenón, Paula; Lajmanovich, Rafael C

2013-09-01

In this study, amphibian tadpoles of Hypsiboas pulchellus were exposed to herbicide Liberty®, which contains glufosinate ammonium (GLA), for 48 h to the following concentrations: 0 (control), 3.55, 4.74, 6.32, 8.43, 11.25, 15, 20, 26.6, and 35.5 mg GLA L(-1). Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, as well as swimming capabilities (swimming speed and mean distance) were measured in tadpoles whose concentrations displayed survival rates > 85 %. Our results reveal that sublethal concentrations of GLA significantly inhibited both AChE and BChE activities in tadpoles with respect to the control, showing a concentration-dependent inhibitory effect. The highest inhibition percentages of AChE (50.86%) and BChE (53.02%) were registered in tadpoles exposed to 15 mg GLA L(-1). At this concentration, a significant increase of the swimming speed and mean distance were found in exposed tadpoles with respect to the control, as well as a negative and significant correlation between swimming speed and BChE activity, thus suggesting that this enzyme inhibition is related to an increase in swimming speed. Therefore, exposure of tadpoles to GLA in the wild at concentrations similar to those tested here may have adverse consequences at population level because neurotransmission and swimming performance are essential for tadpole performance and survival.

Inhibition of acetylcholinesterase and cytochrome oxidase activity in Fasciola gigantica cercaria by phytoconstituents.

PubMed

Sunita, Kumari; Habib, Maria; Kumar, P; Singh, Vinay Kumar; Husain, Syed Akhtar; Singh, D K

2016-02-01

Fasciolosis is an important cattle and human disease caused by Fasciola hepatica and Fasciola gigantica. One of the possible methods to control this problem is to interrupt the life cycle of Fasciola by killing its larva (redia and cercaria) in host snail. Molecular identification of cercaria larva of F. gigantica was done by comparing the nucleotide sequencing with adult F. gigantica. It was noted that nucleotide sequencing of cercaria larva and adult F. gigantica were 99% same. Every month during the year 2011-2012, in vivo treatment with 60% of 4 h LC50 of phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larva of F. gigantica. Whereas, activity of both enzymes were not significantly altered in the nervous tissues of vector snail Lymnaea acuminata exposed to same treatments. Maximum reduction in AChE (1.35% of control in month of June) and cytochrome oxidase (3.71% of control in the month of July) activity were noted in the cercaria exposed to 60% of 4 h LC50 of azadirachtin and allicin, respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

An appraisal of the enzyme stability-activity trade-off.

PubMed

Miller, Scott R

2017-07-01

A longstanding idea in evolutionary physiology is that an enzyme cannot jointly optimize performance at both high and low temperatures due to a trade-off between stability and activity. Although a stability-activity trade-off has been observed for well-characterized examples, such a trade-off is not imposed by any physical chemical constraint. To better understand the pervasiveness of this trade-off, I investigated the stability-activity relationship for comparative biochemical studies of purified orthologous enzymes identified by a literature search. The nature of this relationship varied greatly among studies. Notably, studies of enzymes with low mean synonymous nucleotide sequence divergence were less likely to exhibit the predicted negative correlation between stability and activity. Similarly, a survey of directed evolution investigations of the stability-activity relationship indicated that these traits are often uncoupled among nearly identical yet phenotypically divergent enzymes. This suggests that the presumptive trade-off often reported for investigations of enzymes with high mean sequence divergence may in some cases instead be a consequence of the degeneration over time of enzyme function in unselected environments, rather than a direct effect of thermal adaptation. The results caution against the general assertion of a stability-activity trade-off during enzyme adaptation. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

Spinal activation of alpha7-nicotinic acetylcholine receptor attenuates posttraumatic stress disorder-related chronic pain via suppression of glial activation.

PubMed

Sun, Rao; Zhang, Wei; Bo, Jinhua; Zhang, Zuoxia; Lei, Yishan; Huo, Wenwen; Liu, Yue; Ma, Zhengliang; Gu, Xiaoping

2017-03-06

The high prevalence of chronic pain in posttraumatic stress disorder (PTSD) individuals has been widely reported by clinical studies, which emphasized an urgent need to uncover the underlying mechanisms and identify potential therapeutic targets. Recent studies suggested that targeting activated glia and their pro-inflammatory products may provide a novel and effective therapy for the stress-related pain. In this study, we investigated whether activation of alpha-7 nicotinic acetylcholine receptor (α7 nAChR), a novel anti-inflammatory target, could attenuate PTSD-related chronic pain. The experiments were conducted in a rat model of single prolonged stress (SPS), an established model of PTSD-pain comorbidity. We found that SPS exposure produced persistent mechanical allodynia. Immunohistochemical and enzyme-linked immuno sorbent assay analysis showed that SPS also induced elevated activation of glia cells (including microglia and astrocytes) and accumulation of pro-inflammatory cytokines in spinal cord. In another experiment, we found that intrathecal injection of PHA-543613, a selective α7 nAchR agonist, attenuated the SPS-evoked allodynia in a dose dependent manner. However, this anti-hyperalgesic effect was blocked by pretreatment with methyllycaconitine (MLA), a selective α7 nAchR antagonist. Further analyses showed that PHA-543613 suppressed SPS-induced spinal glial activation and SPS-elevated spinal pro-inflammatory cytokines, and these were abolished by MLA. Taken together, the present study showed that spinal activation of α7 nAChR by PHA-543613 attenuated mechanical allodynia induced by PTSD-like stress, and the suppression of spinal glial activation may underlie this anti-hyperalgesic effect. Our study demonstrated the therapeutic potential of targeting α7 nAChR in the treatment of PTSD-related chronic pain. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Cholinesterase inhibitory activity of isoquinoline alkaloids from three Cryptocarya species (Lauraceae).

PubMed

Wan Othman, Wan Nurul Nazneem; Liew, Sook Yee; Khaw, Kooi Yeong; Murugaiyah, Vikneswaran; Litaudon, Marc; Awang, Khalijah

2016-09-15

Alzheimer's disease is the most common form of dementia among older adults. Acetylcholinesterase and butyrylcholinesterase are two enzymes involved in the breaking down of the neurotransmitter acetylcholine. Inhibitors for these enzymes have potential to prolong the availability of acetylcholine. Hence, the search for such inhibitors especially from natural products is needed in developing potential drugs for Alzheimer's disease. The present study investigates the cholinesterase inhibitory activity of compounds isolated from three Cryptocarya species towards acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Nine alkaloids were isolated; (+)-nornantenine 1, (-)-desmethylsecoantofine 2, (+)-oridine 3, (+)-laurotetanine 4 from the leaves of Cryptocarya densiflora BI., atherosperminine 5, (+)-N-methylisococlaurine 6, (+)-N-methyllaurotetanine 7 from the bark of Cryptocarya infectoria Miq., 2-methoxyatherosperminine 8 and (+)-reticuline 9 from the bark of Cryptocarya griffithiana Wight. In general, most of the alkaloids showed higher inhibition towards BChE as compared to AChE. The phenanthrene type alkaloid; 2-methoxyatherosperminine 8, exhibited the most potent inhibition against BChE with IC50 value of 3.95μM. Analysis of the Lineweaver-Burk (LB) plot of BChE activity over a range of substrate concentration suggested that 2-methoxyatherosperminine 8 exhibited mixed-mode inhibition with an inhibition constant (Ki) of 6.72μM. Molecular docking studies revealed that 2-methoxyatherosperminine 8 docked well at the choline binding site and catalytic triad of hBChE (butyrylcholinesterase from Homo sapiens); hydrogen bonding with Tyr 128 and His 438 residues respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vitro antibody-enzyme conjugates with specific bactericidal activity.

PubMed

Knowles, D M; Sulivan, T J; Parker, C W; Williams, R C

1973-06-01

IgG with antibacterial antibody opsonic activity was isolated from rabbit antisera produced by intravenous hyperimmunization with several test strains of pneumococci, Group A beta-hemolytic streptococci, Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli. Antibody-enzyme conjugates were prepared, using diethylmalonimidate to couple glucose oxidase to IgG antibacterial antibody preparations. Opsonic human IgG obtained from serum of patients with subacute bacterial endocarditis was also conjugated to glucose oxidase. Antibody-enzyme conjugates retained combining specificity for test bacteria as demonstrated by indirect immunofluorescence. In vitro test for bactericidal activity of antibody-enzyme conjugates utilized potassium iodide, lactoperoxidase, and glucose as cofactors. Under these conditions glucose oxidase conjugated to antibody generates hydrogen peroxide, and lactoperoxidase enzyme catalyzes the reduction of hydrogen peroxide with simultaneous oxidation of I(-) and halogenation and killing of test bacteria. Potent in vitro bactericidal activity of this system was repeatedly demonstrated for antibody-enzyme conjugates against pneumococci, streptococci, S. aureus, P. mirabilis, and E. coli. However, no bactericidal effect was demonstrable with antibody-enzyme conjugates and two test strains of P. aeruginosa. Bactericidal activity of antibody-enzyme conjugates appeared to parallel original opsonic potency of unconjugated IgG preparations. Antibody-enzyme conjugates at concentrations as low as 0.01 mg/ml were capable of intense bactericidal activity producing substantial drops in surviving bacterial counts within 30-60 min after initiation of assay. These in vitro bactericidal systems indicate that the concept of antibacterial antibody-enzyme conjugates may possibly be adaptable as a mechanism for treatment of patients with leukocyte dysfunction or fulminant bacteremia.

Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody

PubMed Central

Morsch, Marco; Reddel, Stephen W; Ghazanfari, Nazanin; Toyka, Klaus V; Phillips, William D

2013-01-01

In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft. PMID:23440963

Enzyme-polymer composites with high biocatalytic activity and stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jungbae; Kosto, Timothy J.; Manimala, Joseph C.

2004-08-22

We have applied vacuum-spraying and electrospinning to incorporate an enzyme into a polymer matrix, creating a novel and highly active biocatalytic composite. As a unique technical approach, enzymes were co-dissolved in toluene with polymers, and the solvent was then rapidly removed by injecting the mixture into a vacuum chamber or by electrospinning. Subsequent crosslinking of the enzyme with glutaraldehyde resulted in stable entrapped enzyme within the polymeric matrices. For example, an amorphous composite of alpha-chymotrypsin and polyethylene showed no significant loss of enzymatic activity in aqueous buffer for one month. Nanofibers of alpha-chymotrypsin and polystyrene also showed no decrease inmore » activity for more than two weeks. The normalized activity of amorphous composite in organic solvents was 3-13 times higher than that of native alpha-chymotrypsin. The activity of nanofibers was 5-7 times higher than that of amorphous composite in aqueous buffer solution. The composites of alpha-chymotrypsin and polymers demonstrate the feasibility of obtaining a wide variety of active and stable biocatalytic materials with many combinations of enzymes and polymers.« less

Design, synthesis, acetylcholinesterase inhibition and larvicidal activity of girgensohnine analogs on Aedes aegypti, vector of dengue fever.

PubMed

Carreño Otero, Aurora L; Vargas Méndez, Leonor Y; Duque L, Jonny E; Kouznetsov, Vladimir V

2014-05-06

Girgensohnine alkaloid was used as a natural model in the design and generation of new alkaloid-like α-aminonitrile series that was completed by the use of SSA-catalyzed Strecker reaction between commercial and inexpensive substituted benzaldehydes, piperidine (pyrrolidine, morpholine and N-methylpiperazine) and acetone cyanohydrin. Calculated ADMETox parameters of the designed analogs revealed their good pharmacokinetic profiles indicating lipophilic characteristics. In vitro AChE enzyme test showed that obtained α-aminonitriles could be considered as AChEIs with micromolar IC50 values ranging from 42.0 to 478.0 μM (10.3-124.0 μg/mL). Among this series, the best AChE inhibitor was the pyrrolidine α-aminonitrile 3 (IC50 = 42 μM), followed by the piperidine α-aminonitriles 2 and 6 (IC50 = 45 μM and IC50 = 51 μM, respectively), and the compound 7 (IC50 = 51 μM). In vivo insecticidal activity of more active AChEIs against Aedes aegypti larvae was also performed showing a good larvicidal activity at concentrations less than 140 ppm, highlighting products 2 and 7 that could serve as lead compounds to develop new potent and selective insecticides. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Gender-related differences in circadian rhythm of rat plasma acetyl- and butyrylcholinesterase: effects of sex hormone withdrawal.

PubMed

Alves-Amaral, Gracielle; Pires-Oliveira, Marcelo; Andrade-Lopes, Ana Luiza; Chiavegatti, Tiago; Godinho, Rosely Oliveira

2010-06-07

The role of acetylcholinesterase (AChE) in the termination of the cholinergic response through acetylcholine (ACh) hydrolysis and the involvement of plasma butyrylcholinesterase (BuChE), mainly of hepatic origin, in the metabolism of xenobiotics with ester bonds is well known. Besides, BuChE has a crucial role in ACh hydrolysis, especially when selective anticholinesterases inhibit AChE. Herein, we analyzed the gender-related differences and the circadian changes of rat plasma cholinesterases. Plasma and liver cholinesterase activities were evaluated in control or 2-30-day castrated adult male and female rats. Plasma and liver AChE activities did not differ between genders and were not influenced by sex hormone deprivation. BuChE plasma activity was 7 times greater in female, reflecting gender differences in liver enzyme expression. Castration increased liver and plasma BuChE activity in male, while reduced it in female, abolishing gender differences in enzyme activity. Interestingly, female AChE and BuChE plasma activities varied throughout the day, reaching values 27% and 42% lower, respectively, between 2 p.m. and 6 p.m. when compared to the morning peaks at 8 a.m. Castration attenuated daily female BuChE oscillation. On the other hand, male plasma enzymes remained constant throughout the day. In summary, our results show that liver and plasma BuChE, but not AChE, expression is influenced by sex hormones, leading to high levels of blood BuChE in females. The fluctuation of female plasma BuChE during the day should be taken into account to adjust the bioavailability and the therapeutic effects of cholinesterase inhibitors used in cholinergic-based conditions such Alzheimer's disease. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

The in vitro comparative study of the effect of BPA, BPS, BPF and BPAF on human erythrocyte membrane; perturbations in membrane fluidity, alterations in conformational state and damage to proteins, changes in ATP level and Na+/K+ ATPase and AChE activities.

PubMed

Maćczak, Aneta; Duchnowicz, Piotr; Sicińska, Paulina; Koter-Michalak, Maria; Bukowska, Bożena; Michałowicz, Jaromir

2017-12-01

Bisphenols are massively used in the industry, and thus the exposure of biota including humans to these substances has been noted. In this study we have assessed the effect of BPA and its selected analogs, i.e. BPS, BPF and BPAF on membrane of human red blood cells, which is the first barrier that must be overcome by xenobiotics penetrating the cell, and is commonly utilized as a model in the investigation of the effect of different xenobiotics on various cell types. Red blood cells were incubated with BPA and its analogs in the concentrations ranging from 0.1 to 250 μg/ml for 4 h and 24 h. We have noted that the compounds studied altered membrane fluidity at its hydrophobic region, increased internal viscosity and osmotic fragility of the erythrocytes and altered conformational state of membrane proteins. Moreover, bisphenols examined increased thiol groups level, caused oxidative damage to membrane proteins, decreased ATP level, depleted the activity of Na+/K + ATPase and changed the activity of AChE in human red blood cells. It has been shown that the strongest changes were noted in cells treated with BPAF, while BPS caused the weakest (or none) alterations in the parameters studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

Deficiency of cellulase activity measurements for enzyme evaluation.

PubMed

Pryor, Scott W; Nahar, Nurun

2010-11-01

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial beta-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.

Modulation of hippocampal ACh release by chronic nicergoline treatment in freely moving young and aged rats.

PubMed

Carfagna, N; Di Clemente, A; Cavanus, S; Damiani, D; Gerna, M; Salmoiraghi, P; Cattaneo, B; Post, C

1995-09-15

The effects of nicergoline on basal and K(+)-stimulated release of ACh in the hippocampus of 3- and 19-month old rats has been studied by microdialysis. A significant decrease of basal ACh release (59%) was found in aged vehicle treated rats in comparison to young rats. High-K+ (100 mM) in the perfusate strongly increased the release of ACh by up to 6-fold over the baseline of both young and aged rats. Chronic oral administration of nicergoline to aged rats (5 mg/kg b.i.d. for 6 weeks) significantly reversed (93%) the age-related decrease of basal release of ACh, leaving the increase due to K+ depolarization unchanged. In young animals, nicergoline did not affect the basal output of ACh, but enhanced the K(+)-evoked release of ACh by 39%. Results from this study demonstrate that nicergoline treatment increases the ability of hippocampal cholinergic terminals to release ACh, and suggest that this drug can reset the cholinergic impairement associated with aging.

Silibinin inhibits acetylcholinesterase activity and amyloid β peptide aggregation: a dual-target drug for the treatment of Alzheimer's disease.

PubMed

Duan, Songwei; Guan, Xiaoyin; Lin, Runxuan; Liu, Xincheng; Yan, Ying; Lin, Ruibang; Zhang, Tianqi; Chen, Xueman; Huang, Jiaqi; Sun, Xicui; Li, Qingqing; Fang, Shaoliang; Xu, Jun; Yao, Zhibin; Gu, Huaiyu

2015-05-01

Alzheimer's disease (AD) is characterized by amyloid β (Aβ) peptide aggregation and cholinergic neurodegeneration. Therefore, in this paper, we examined silibinin, a flavonoid extracted from Silybum marianum, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aβ peptide aggregation for AD treatment. To achieve this, we used molecular docking and molecular dynamics simulations to examine the affinity of silibinin with Aβ and AChE in silico. Next, we used circular dichroism and transmission electron microscopy to study the anti-Aβ aggregation capability of silibinin in vitro. Moreover, a Morris Water Maze test, enzyme-linked immunosorbent assay, immunohistochemistry, 5-bromo-2-deoxyuridine double labeling, and a gene gun experiment were performed on silibinin-treated APP/PS1 transgenic mice. In molecular dynamics simulations, silibinin interacted with Aβ and AChE to form different stable complexes. After the administration of silibinin, AChE activity and Aβ aggregations were down-regulated, and the quantity of AChE also decreased. In addition, silibinin-treated APP/PS1 transgenic mice had greater scores in the Morris Water Maze. Moreover, silibinin could increase the number of newly generated microglia, astrocytes, neurons, and neuronal precursor cells. Taken together, these data suggest that silibinin could act as a dual inhibitor of AChE and Aβ peptide aggregation, therefore suggesting a therapeutic strategy for AD treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

Pharmacological and immunochemical characterization of α2* nicotinic acetylcholine receptors (nAChRs) in mouse brain

PubMed Central

Whiteaker, Paul; Wilking, Jennifer A; Brown, Robert WB; Brennan, Robert J; Collins, Allan C; Lindstrom, Jon M; Boulter, Jim

2009-01-01

Aim: α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2* nAChRs in these mouse brain regions. Methods: α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing (α2*) nAChRs in these two regions. Results: [125I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8±1.7 and 92±26 fmol̇mg-1 protein, respectively). Pharmacological characterization using the β2-selective drug A85380 to inhibit [125I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2* nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good (>90% of β2* and >80% of β4* nAChRs were routinely recovered). Conclusion: Immunoprecipitation experiments indicated that interpeduncular nucleus α2* nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits. PMID:19498420

Investigations into the development of catalytic activity in anti-acetylcholinesterase idiotypic and anti-idiotypic antibodies.

PubMed

Johnson, Glynis; Moore, Samuel W

2009-01-01

We have previously described anti-acetylcholinesterase antibodies that display acetylcholinesterase-like catalytic activity. No evidence of contaminating enzymes was found, and the antibodies are kinetically and apparently structurally distinct from both acetylcholinesterase (AChE) and butyrylcholinesterase. We have also mimicked the antibody catalytic sites in anti-anti-idiotypic (Ab3) antibodies. Independently from us, similar acetylcholinesterase-like antibodies have been raised as anti-idiotypic (Ab2) antibodies against a non-catalytic anti-acetylcholinesterase antibody, AE-2. In this paper, we describe an epitope analysis, using synthetic peptides in ELISA and competition ELISA, and a peptide array, of five catalytic anti-acetylcholinesterase antibodies (Ab1s), three catalytic Ab3s, as well as antibody AE-2 and a non-catalytic Ab2. The catalytic Ab1s and Ab3s recognized three Pro- and Gly-containing sequences ((40)PPMGPRRFL, (78)PGFEGTE, and (258)PPGGTGGNDTELVAC) on the AChE surface. As these sequences do not adjoin in the AChE structure, recognition would appear to be due to cross-reaction. This was confirmed by the observation that the sequences superimpose structurally. The non-catalytic antibodies, AE-2 and the Ab2, recognized AChE's peripheral anionic site (PAS), in particular, the sequence (70)YQYVD, which contains two of the site's residues. The crystal structure of the AChE tetramer (Bourne et al., 1999) shows direct interaction and high complementarity between the (257)CPPGGTGGNDTELVAC sequence and the PAS. Antibodies recognizing the sequence and the PAS may, in turn, be complementary; this may account for the apparent paradox of catalytic development in both Ab1s and Ab2s. The PAS binds, but does not hydrolyze, substrate. The catalytic Ab1s, therefore, recognize a site that may function as a substrate analog, and this, together with the presence of an Arg-Glu salt bridge in the epitope, suggests mechanisms whereby catalytic activity may have

Synthesis, biological evaluation and docking studies of 2,3-dihydroquinazolin-4(1H)-one derivatives as inhibitors of cholinesterases.

PubMed

Sarfraz, Muhammad; Sultana, Nargis; Rashid, Umer; Akram, Muhammad Safwan; Sadiq, Abdul; Tariq, Muhammad Ilyas

2017-02-01

In search of potent inhibitors of cholinesterases, we have synthesized and evaluate a number of 2,3-dihydroquinazolin-4(1H)-one derivatives. The synthetic approach provided an efficient synthesis of the target molecules with excellent yield. All the tested compounds showed activity against both the enzymes in micromolar range. In many case, the inhibition of both enzymes are higher than or comparable to the standard drug galatamine. With the selectivity index of 2.3 for AChE, compound 5f can be considered as a potential lead compound with a feature of dual AChE/BChE inhibition with IC 50 =1.6±0.10μM (AChE) and 3.7±0.18μM (BChE). Binding modes of the synthesized compounds were explored by using GOLD (Genetic Optimization for Ligand Docking) suit v5.4.1. The computed binding modes of these compounds in the active site of AChE and BChE provide an insight into the mechanism of inhibition of these two enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, Pratul K

2012-01-01

Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted thatmore » mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.« less

Low dielectric response in enzyme active site

PubMed Central

Mertz, Edward L.; Krishtalik, Lev I.

2000-01-01

The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

Cholinesterase Inhibitory Activity of Some semi-Rigid Spiro Heterocycles: POM Analyses and Crystalline Structure of Pharmacophore Site.

PubMed

Hadda, Taibi Ben; Talhi, Oualid; Silva, Artur S M; Senol, Fatma Sezer; Orhan, Ilkay Erdogan; Rauf, Abdur; Mabkhot, Yahia N; Bachari, Khaldoun; Warad, Ismail; Farghaly, Thoraya A; Althagafi, Ismail I; Mubarak, Mohammad S

2018-01-01

Cholinesterase family consists of two sister enzymes; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which hydrolyze acetylcholine. Since deficit of acetylcholine has been evidenced in patients of Alzheimer's disease (AD), cholinesterase inhibitors are currently the most prescribed drugs for the treatment of AD. our aim in this article was to investigate the inhibitory potential of five known compounds (2-6) with spiro skeleton against AChE and BChE using ELISA microplate assays. In addition to their ChE inhibitory effect, their physico-chemical properties were also calculated. Moreover, the present work aims at investigating the charge/geometrical effect of a hypothetical pharmacophore or bidentate site in a bioactive group, on the inhibition efficiency of spiro compounds 2-6 by using Petra/Osiris/ molinspiration (POM) and X-ray analyses. In the present study, five compounds (2-6) with spiro skeleton have been synthesized and tested in vitro for their inhibitory potential against AChE and BChE using ELISA microtiter plate assays at 25 µg/mL. Results revealed that three of the spiro compounds tested exert more than 50% inhibition against one of cholinesterases. Compound 5 displayed 68.73 ± 4.73% of inhibition toward AChE, whereas compound 6 showed 56.17 ± 0.83% of inhibition toward BChE; these two previously synthesized compounds have been the most active hits. Our data obtained from screening of compounds 2-6 against the two cholinesterases indicate that three of these show good potential to selectively inhibit AChE or BChE. Spiro compounds 2, 5, and 6 exhibited the most potent activity of the series against AChE or BChE with inhibition values in the range 55-70%. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Inhibition of existing denitrification enzyme activity by chloramphenicol

USGS Publications Warehouse

Brooks, M.H.; Smith, R.L.; Macalady, D.L.

1992-01-01

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

Dysregulated Homeostasis of Acetylcholine Levels in Immune Cells of RR-Multiple Sclerosis Patients.

PubMed

Di Bari, Maria; Reale, Marcella; Di Nicola, Marta; Orlando, Viviana; Galizia, Sabrina; Porfilio, Italo; Costantini, Erica; D'Angelo, Chiara; Ruggieri, Serena; Biagioni, Stefano; Gasperini, Claudio; Tata, Ada Maria

2016-11-30

Multiple sclerosis (MS) is characterized by pro-inflammatory cytokine production. Acetylcholine (ACh) contributes to the modulation of central and peripheral inflammation. We studied the homeostasis of the cholinergic system in relation to cytokine levels in immune cells and sera of relapsing remitting-MS (RR-MS) patients. We demonstrated that lower ACh levels in serum of RR-MS patients were inversely correlated with the increased activity of the hydrolyzing enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Interestingly, the expression of the ACh biosynthetic enzyme and the protein carriers involved in non-vesicular ACh release were found overexpressed in peripheral blood mononuclear cells of MS patients. The inflammatory state of the MS patients was confirmed by increased levels of TNFα, IL-12/IL-23p40, IL-18. The lower circulating ACh levels in sera of MS patients are dependent on the higher activity of cholinergic hydrolyzing enzymes. The smaller ratio of ACh to TNFα, IL-12/IL-23p40 and IL-18 in MS patients, with respect to healthy donors (HD), is indicative of an inflammatory environment probably related to the alteration of cholinergic system homeostasis.

Dysregulated Homeostasis of Acetylcholine Levels in Immune Cells of RR-Multiple Sclerosis Patients

PubMed Central

Di Bari, Maria; Reale, Marcella; Di Nicola, Marta; Orlando, Viviana; Galizia, Sabrina; Porfilio, Italo; Costantini, Erica; D’Angelo, Chiara; Ruggieri, Serena; Biagioni, Stefano; Gasperini, Claudio; Tata, Ada Maria

2016-01-01

Multiple sclerosis (MS) is characterized by pro-inflammatory cytokine production. Acetylcholine (ACh) contributes to the modulation of central and peripheral inflammation. We studied the homeostasis of the cholinergic system in relation to cytokine levels in immune cells and sera of relapsing remitting-MS (RR-MS) patients. We demonstrated that lower ACh levels in serum of RR-MS patients were inversely correlated with the increased activity of the hydrolyzing enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Interestingly, the expression of the ACh biosynthetic enzyme and the protein carriers involved in non-vesicular ACh release were found overexpressed in peripheral blood mononuclear cells of MS patients. The inflammatory state of the MS patients was confirmed by increased levels of TNFα, IL-12/IL-23p40, IL-18. The lower circulating ACh levels in sera of MS patients are dependent on the higher activity of cholinergic hydrolyzing enzymes. The smaller ratio of ACh to TNFα, IL-12/IL-23p40 and IL-18 in MS patients, with respect to healthy donors (HD), is indicative of an inflammatory environment probably related to the alteration of cholinergic system homeostasis. PMID:27916909

In vivo pharmacological interactions between a type II positive allosteric modulator of α7 nicotinic ACh receptors and nicotinic agonists in a murine tonic pain model

PubMed Central

Freitas, K; Negus, SS; Carroll, FI; Damaj, MI

2013-01-01

Background and Purpose The α7 nicotinic ACh receptor subtype is abundantly expressed in the CNS and in the periphery. Recent evidence suggests that α7 nicotinic ACh receptor (nAChR) subtypes, which can be activated by an endogenous cholinergic tone comprising ACh and the α7 agonist choline, play an important role in chronic pain and inflammation. In this study, we evaluated whether type II α7 positive allosteric modulator PNU-120596 induces antinociception on its own and in combination with choline in the formalin pain model. Experimental Approach We assessed the effects of PNU-120596 and choline and the nature of their interactions in the formalin test using an isobolographic analysis. In addition, we evaluated the interaction of PNU-120596 with PHA-54613, an exogenous selective α7 nAChR agonist, in the formalin test. Finally, we assessed the interaction between PNU-120596 and nicotine using acute thermal pain, locomotor activity, body temperature and convulsing activity tests in mice. Key Results We found that PNU-120596 dose-dependently attenuated nociceptive behaviour in the formalin test after systemic administration in mice. In addition, mixtures of PNU-120596 and choline synergistically reduced formalin-induced pain. PNU-120596 enhanced the effects of nicotine and α7 agonist PHA-543613 in the same test. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, hypomotility and antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via activation of α7 nAChRs. Conclusions and Implications Our results demonstrate that type II α7 positive allosteric modulators produce antinociceptive effects in the formalin test through a synergistic interaction with the endogenous α7 agonist choline. PMID:23004024

Diffusional correlations among multiple active sites in a single enzyme.

PubMed

Echeverria, Carlos; Kapral, Raymond

2014-04-07

Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

Can Salivary Acetylcholinesterase be a Diagnostic Biomarker for Alzheimer?

PubMed

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

2017-01-01

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. 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. 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. 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). 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.

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle.

PubMed

Franco, L; Bruzzone, S; Song, P; Guida, L; Zocchi, E; Walseth, T F; Crimi, E; Usai, C; De Flora, A; Brusasco, V

2001-01-01

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.

Perspectives on the Use of Scopolamine as an Adjunct Treatment to Enhance Survival Following Organophosphorus Nerve Agent Poisoning

DTIC Science & Technology

2010-11-01

particular nerve agent and oxime uti- lized in the treatment regimen. Atropine is the universal treatment for organophospho- rus anticholinesterase poisoning...general, to the recovery of AChE activity either through decarbamylation of PB protected enzyme or by use of an effective oxime. The results against...this protected enzyme in the first few minutes after intoxication and treat- ment provides sufficient enzyme activity to sustain survival.̂ ’"* A

Physics-based enzyme design: predicting binding affinity and catalytic activity.

PubMed

Sirin, Sarah; Pearlman, David A; Sherman, Woody

2014-12-01

Computational enzyme design is an emerging field that has yielded promising success stories, but where numerous challenges remain. Accurate methods to rapidly evaluate possible enzyme design variants could provide significant value when combined with experimental efforts by reducing the number of variants needed to be synthesized and speeding the time to reach the desired endpoint of the design. To that end, extending our computational methods to model the fundamental physical-chemical principles that regulate activity in a protocol that is automated and accessible to a broad population of enzyme design researchers is essential. Here, we apply a physics-based implicit solvent MM-GBSA scoring approach to enzyme design and benchmark the computational predictions against experimentally determined activities. Specifically, we evaluate the ability of MM-GBSA to predict changes in affinity for a steroid binder protein, catalytic turnover for a Kemp eliminase, and catalytic activity for α-Gliadin peptidase variants. Using the enzyme design framework developed here, we accurately rank the most experimentally active enzyme variants, suggesting that this approach could provide enrichment of active variants in real-world enzyme design applications. © 2014 Wiley Periodicals, Inc.

Regulated Extracellular Choline Acetyltransferase Activity— The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway

PubMed Central

Aeinehband, Shahin; Behbahani, Homira; Grandien, Alf; Nilsson, Bo; Ekdahl, Kristina N.; Lindblom, Rickard P. F.; Piehl, Fredrik; Darreh-Shori, Taher

2013-01-01

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer’s disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states

Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates

NASA Astrophysics Data System (ADS)

Fuhrmann, Gregor; Grotzky, Andrea; Lukić, Ružica; Matoori, Simon; Luciani, Paola; Yu, Hao; Zhang, Baozhong; Walde, Peter; Schlüter, A. Dieter; Gauthier, Marc A.; Leroux, Jean-Christophe

2013-07-01

Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

Sympathetic α₃β₂-nAChRs mediate cerebral neurogenic nitrergic vasodilation in the swine.

PubMed

Lee, Reggie Hui-Chao; Liu, Yi-Qing; Chen, Po-Yi; Liu, Chin-Hung; Chen, Mei-Fang; Lin, Hung-Wen; Kuo, Jon-Son; Premkumar, Louis S; Lee, Tony Jer-Fu

2011-08-01

The α(7)-nicotinic ACh receptor (α(7)-nAChR) on sympathetic neurons innervating basilar arteries of pigs crossed bred between Landrace and Yorkshire (LY) is known to mediate nicotine-induced, β-amyloid (Aβ)-sensitive nitrergic neurogenic vasodilation. Preliminary studies, however, demonstrated that nicotine-induced cerebral vasodilation in pigs crossbred among Landrace, Yorkshire, and Duroc (LYD) was insensitive to Aβ and α-bungarotoxin (α-BGTX). We investigated nAChR subtype on sympathetic neurons innervating LYD basilar arteries. Nicotine-induced relaxation of porcine isolated basilar arteries was examined by tissue bath myography, inward currents on nAChR-expressing oocytes by two-electrode voltage recording, and mRNA and protein expression in the superior cervical ganglion (SCG) and middle cervical ganglion (MCG) by reverse transcription PCR and Western blotting. Nicotine-induced basilar arterial relaxation was not affected by Aβ, α-BGTX, and α-conotoxin IMI (α(7)-nAChR antagonists), or α-conotoxin AuIB (α(3)β(4)-nAChR antagonist) but was inhibited by tropinone and tropane (α(3)-containing nAChR antagonists) and α-conotoxin MII (selective α(3)β(2)-nAChR antagonist). Nicotine-induced inward currents in α(3)β(2)-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aβ, or α-conotoxin AuIB. mRNAs of α(3)-, α(7)-, β(2)-, and β(4)-subunits were expressed in both SCGs and MCGs with significantly higher mRNAs of α(3)-, β(2)-, and β(4)-subunits than that of α(7)-subunit. The Aβ-insensitive sympathetic α(3)β(2)-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aβ-sensitive α(7)-nAChR in basilar arteries of LY pigs may offer a partial explanation for different sensitivities of individuals to Aβ in causing diminished cerebral nitrergic vasodilation in diseases involving Aβ.

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...

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

PubMed

Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

NASA Astrophysics Data System (ADS)

Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

APS8, a Polymeric Alkylpyridinium Salt Blocks α7 nAChR and Induces Apoptosis in Non-Small Cell Lung Carcinoma

PubMed Central

Zovko, Ana; Viktorsson, Kristina; Lewensohn, Rolf; Kološa, Katja; Filipič, Metka; Xing, Hong; Kem, William R.; Paleari, Laura; Turk, Tom

2013-01-01

Naturally occurring 3-alkylpyridinium polymers (poly-APS) from the marine sponge Reniera sarai, consisting of monomers containing polar pyridinium and nonpolar alkyl chain moieties, have been demonstrated to exert a wide range of biological activities, including a selective cytotoxicity against non-small cell lung cancer (NSCLC) cells. APS8, an analog of poly-APS with defined alkyl chain length and molecular size, non-competitively inhibits α7 nicotinic acetylcholine receptors (nAChRs) at nanomolar concentrations that are too low to be acetylcholinesterase (AChE) inhibitory or generally cytotoxic. In the present study we show that APS8 inhibits NSCLC tumor cell growth and activates apoptotic pathways. APS8 was not toxic for normal lung fibroblasts. Furthermore, in NSCLC cells, APS8 reduced the adverse anti-apoptotic, proliferative effects of nicotine. Our results suggest that APS8 or similar compounds might be considered as lead compounds to develop antitumor therapeutic agents for at least certain types of lung cancer. PMID:23880932

Characterization of Soil Samples of Enzyme Activity

ERIC Educational Resources Information Center

Freeland, P. W.

1977-01-01

Described are nine enzyme essays for distinguishing soil samples. Colorimetric methods are used to compare enzyme levels in soils from different sites. Each soil tested had its own spectrum of activity. Attention is drawn to applications of this technique in forensic science and in studies of soil fertility. (Author/AJ)

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

The molecular basis of the effect of temperature on enzyme activity.

PubMed

Daniel, Roy M; Peterson, Michelle E; Danson, Michael J; Price, Nicholas C; Kelly, Sharon M; Monk, Colin R; Weinberg, Cristina S; Oudshoorn, Matthew L; Lee, Charles K

2009-12-23

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

Ionizable Side Chains at Catalytic Active Sites of Enzymes

PubMed Central

Jimenez-Morales, David; Liang, Jie

2012-01-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

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.

Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

NASA Astrophysics Data System (ADS)

Allison, S. D.; Jastrow, J. D.

2004-12-01

Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

Function and biotechnology of extremophilic enzymes in low water activity

PubMed Central

2012-01-01

Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology. PMID:22480329

H2 formation via the UV photo-processing of a-C:H nano-particles

NASA Astrophysics Data System (ADS)

Jones, A. P.; Habart, E.

2015-09-01

Context. The photolysis of hydrogenated amorphous carbon, a-C(:H), dust by UV photon-irradiation in the laboratory leads to the release of H2 as well as other molecules and radicals. This same process is also likely to be important in the interstellar medium. Aims: We investigate molecule formation arising from the photo-dissociatively-driven, regenerative processing of a-C(:H) dust. Methods: We explore the mechanism of a-C(:H) grain photolysis leading to the formation of H2 and other molecules/radicals. Results: The rate constant for the photon-driven formation of H2 from a-C(:H) grains is estimated to be 2 × 10-17 cm3 s-1. In intense radiation fields photon-driven grain decomposition will lead to fragmentation into daughter species rather than H2 formation. Conclusions: The cyclic re-structuring of arophatic a-C(:H) nano-particles appears to be a viable route to formation of H2 for low to moderate radiation field intensities (1 ≲ G0 ≲ 102), even when the dust is warm (T ~ 50-100 K).

ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

PubMed Central

Stine, G. J.

1968-01-01

Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

Erythrocyte acetylcholinesterase as biomarker of pesticide exposure: new and forgotten insights.

PubMed

Assis, Caio R D; Linhares, Amanda G; Cabrera, Mariana P; Oliveira, Vagne M; Silva, Kaline C C; Marcuschi, Marina; Maciel Carvalho, Elba V M; Bezerra, Ranilson S; Carvalho, Luiz B

2018-05-24

Acetylcholinesterase (AChE) acts on the hydrolysis of acetylcholine, rapidly removing this neurotransmitter at cholinergic synapses and neuromuscular junctions as well as in neuronal growth and differentiation, modulation of cell adhesion ("electrotactins") and aryl-acylamidase activity (AAA). This enzyme is also found in erythrocyte, as 160 kDa dimer that anchors to the plasma membrane via glycophosphatidylinositol. The function of this enzyme in erythrocytes has not yet been elucidated; however, it is suspected to participate in cell-to-cell interactions. Here, a review on erythrocyte AChE characteristics and use as biomarker for organophosphorus and carbamate insecticides is presented since it is the first specific target/barrier of the action of these pesticides, besides plasma butyrylcholinesterase (BChE). However, some past and current methods have disadvantages: (a) not discriminating the activities of AChE and BChE; (b) low accuracy due to interference of hemoglobin in whole blood samples. On the other hand, extraction methods of hemoglobin-free erythrocyte AChE allows: (a) the freezing and transporting of samples; (b) samples free of colorimetric interference; (c) data from only erythrocyte AChE activity; (d) erythrocyte AChE specific activity presents higher correlation with the central nervous system AChE than other peripheral ChEs; (e) slow spontaneous regeneration against anti-ChEs agents of AChE in comparison to BChE, thus increasing the chances of detecting such compounds following longer interval after exposure. As monitoring perspectives, hemoglobin-free methodologies may be promising alternatives to assess the degree of exposure since they are not influenced by this interfering agent.

Molecular modeling and structural analysis of nAChR variants uncovers the mechanism of resistance to snake toxins.

PubMed

Gunasekaran, D; Sridhar, J; Suryanarayanan, V; Manimaran, N C; Singh, Sanjeev Kumar

2017-06-01

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.

Synthesis of the signal molecule acetylcholine during the developmental cycle of Paramecium primaurelia (Protista, Ciliophora) and its possible function in conjugation.

PubMed

Delmonte Corrado, M U; Politi, H; Ognibene, M; Angelini, C; Trielli, F; Ballarini, P; Falugi, C

2001-06-01

We recently discovered, in mating-competent Paramecium primaurelia, the presence of functionally related molecules of the cholinergic system: the neurotransmitter acetylcholine (ACh), both its nicotinic and muscarinic receptors and its lytic enzyme acetylcholinesterase (AChE). Our results on the inhibition of mating-cell pairing in vivo in mating-competent cells treated with cholinomimetic drugs support the hypothesis that the cholinergic system plays a role in cell-to-cell adhesion. To investigate the possible function of the signal molecule ACh in conjugation in P. primaurelia, we attempted to detect the intracellular sites of ACh synthesis by localizing the ACh biosynthetic enzyme choline acetyltransferase (ChAT). Using immunocytochemical and histochemical methods, we have demonstrated the presence and activity of ChAT principally on the surface membrane of mating-competent cells and of mature but non-mating-competent cells. No evidence for ChAT activity was found in immature cells. Immunoblot analysis revealed the presence of immunoreactive bands, ranging in molecular mass from 42 to 133 kDa, as reported for ChAT isolated from higher organisms. In vivo experiments showed that inhibition of ChAT activity by Congo Red, known to be a potent competitive inhibitor of acetyl coenzyme A, did not affect mating-cell pairing. Conversely, inhibition of AChE with BW 284c51 or eserine, which block enzyme activity by reacting with a specific serine within the catalytic centre, significantly inhibited mating-cell pairing. Our results suggest that ACh has a negative modulating effect on conjugation in P. primaurelia.

Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

PubMed

Pavelka, S

2014-01-01

We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between

Anti-cholinesterase activity of the standardized extract of Syzygium aromaticum L.

PubMed

Dalai, Manoj K; Bhadra, Santanu; Chaudhary, Sushil K; Bandyopadhyay, Arun; Mukherjee, Pulok K

2014-04-01

Clove (Syzygium aromaticum) is a well-known culinary spice with strong aroma; contains a high amount of oil known as clove oil. The major phyto-constituent of the clove oil is eugenol. Clove and its oil possess various medicinal uses in indigenous medicine as an antiseptic, anti-oxidant, analgesic and neuroprotective properties. Thus, it draws much attention among researchers from pharmaceutical, food and cosmetic industries. The aim of the present study was to determine the anti-cholinesterase activity of the methanol extract of clove, its oil and eugenol. In vitro anti-cholinesterase activity of S. aromaticum was performed by a thin layer chromatography bio autography, 96 well micro titer plate and kinetic methods. Reverse phase high performance liquid chromatography (RP-HPLC) analysis was carried out to identify the biomarker compound eugenol in clove oil. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition study revealed that eugenol possess better inhibition of the enzymes than extract and oil. Clove extract, its oil and eugenol showed better inhibition of AChE than BChE. Polyphenolic compound eugenol was detected through RP-HPLC analysis. The content of eugenol in essential oil was found to be 0.5 μg/ml. Kinetic analysis of the cholinesterase inhibition study of the extract; clove oil and eugenol have shown that they possess mixed type of inhibition for AChE and non-competitive type of inhibition for BChE. These results might be useful in explaining the effect of clove as anti-cholinesterase agent for the management of cognitive ailments like Alzheimer's disease.

α5 nAChR modulation of the prefrontal cortex makes attention resilient.

PubMed

Howe, William M; Brooks, Julie L; Tierney, Patrick L; Pang, Jincheng; Rossi, Amie; Young, Damon; Dlugolenski, Keith; Guillmette, Ed; Roy, Marc; Hales, Katherine; Kozak, Rouba

2018-03-01

A loss-of-function polymorphism in the α5 nicotinic acetylcholine receptor (nAChR) subunit gene has been linked to both drug abuse and schizophrenia. The α5 nAChR subunit is strategically positioned in the prefrontal cortex (PFC), where a loss-of-function in this subunit may contribute to cognitive disruptions in both disorders. However, the specific contribution of α5 to PFC-dependent cognitive functions has yet to be illustrated. In the present studies, we used RNA interference to knockdown the α5 nAChR subunit in the PFC of adult rats. We provide evidence that through its contribution to cholinergic modulation of cholinergic modulation of neurons in the PFC, the α5 nAChR plays a specific role in the recovery of attention task performance following distraction. Our combined data reveal the potent ability of this subunit to modulate the PFC and cognitive functions controlled by this brain region that are impaired in disease.

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity[S

PubMed Central

Jaishy, Bharat; Zhang, Quanjiang; Chung, Heaseung S.; Riehle, Christian; Soto, Jamie; Jenkins, Stephen; Abel, Patrick; Cowart, L. Ashley; Van Eyk, Jennifer E.; Abel, E. Dale

2015-01-01

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs. PMID:25529920

Ionizable side chains at catalytic active sites of enzymes.

PubMed

Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

2012-05-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

Distribution of enzyme activity hotspots induced by earthworms in top- and subsoil

NASA Astrophysics Data System (ADS)

Hoang, D. T. T.

2016-12-01

Earthworms (Lumbricus terrestris L.) not only affect soil physics, but they also boost microbial activities and consequently create important hotspots of microbial mediated carbon and nutrient turnover through their burrowing activity. However, it is still unknown to which extend earthworms change the enzyme distribution and activity inside their burrows in top- and subsoil horizons. We hypothesized that earthworm burrows, which are enriched in available substrates, have higher percentage of enzyme activity hotspots than soil without earthworms, and that enzyme activities decreased with increasing depth because of the increasing recalcitrance of organic matter in subsoil. We visualized enzyme distribution inside and outside of worm burrows (biopores) by in situ soil zymography and measured enzyme kinetics of 6 enzymes - β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) - in pore and bulk soil material up to 105 cm. Zymography showed a heterogeneous distribution of hotspots in worm burrows. The hotspot areas was 2.4 to 14 times larger in the burrows than in soil without earthworms. However, the dispersion index of hotspot distribution showed more aggregated hotspots in soil without earthworms than in soil with earthworms and burrow wall. Enzyme activities decreased with depth, by a factor of 2 to 8 due to fresh C input from the soil surface. Compared to bulk soil, enzyme activities in topsoil biopores were up to 11 times higher for all enzymes, but in the subsoil activities of XYL, NAG and APT were lower in earthworm biopores than bulk soil. In conclusion, hotspots were twice as concentrated close to earthworm burrows as in surrounding soil. Earthworms exerted stronger effects on enzyme activities in biopores in the topsoil than in subsoil. Keywords: Earthworms, hotspots, enzyme activities, enzyme distribution, subsoil

Kinetics and Molecular Docking Studies of 6-Formyl Umbelliferone Isolated from Angelica decursiva as an Inhibitor of Cholinesterase and BACE1.

PubMed

Ali, Md Yousof; Seong, Su Hui; Reddy, Machireddy Rajeshkumar; Seo, Sung Yong; Choi, Jae Sue; Jung, Hyun Ah

2017-09-24

Coumarins, which have low toxicity, are present in some natural foods, and are used in various herbal remedies, have attracted interest in recent years because of their potential medicinal properties. In this study, we report the isolation of two natural coumarins, namely umbelliferone ( 1 ) and 6-formyl umbelliferone ( 2 ), from Angelica decursiva , and the synthesis of 8-formyl umbelliferone ( 3 ) from 1 . We investigated the anti-Alzheimer disease (anti-AD) potential of these coumarins by assessing their ability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1). Among these coumarins, 2 exhibited poor inhibitory activity against AChE and BChE, and modest activity against BACE1. Structure-activity relationship analysis showed that 2 has an aldehyde group at the C-6 position, and exhibited strong anti-AD activity, whereas the presence or absence of an aldehyde group at the C-8 position reduced the anti-AD activity of 3 and 1 , respectively. In addition, 2 exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. A kinetic study revealed that 2 and 3 non-competitively inhibited BACE1. To confirm enzyme inhibition, we predicted the 3D structures of AChE and BACE1, and used AutoDock 4.2 to simulate binding of coumarins to these enzymes. The blind docking studies demonstrated that these molecules could interact with both the catalytic active sites and peripheral anionic sites of AChE and BACE1. Together, our results indicate that 2 has an interesting inhibitory activity in vitro, and can be used in further studies to develop therapeutic modalities for the treatment of AD.

Single channel properties of human α3 AChRs: impact of β2, β4 and α5 subunits

PubMed Central

Nelson, Mark E; Lindstrom, Jon

1999-01-01

We performed single channel analysis on human α3 acetylcholine receptors (AChRs) in Xenopus oocytes and native AChRs from the human neuroblastoma cell line IMR-32. α3 AChRs exhibit channel properties that reflect subunit composition.α3β2 AChR open times were 0.71 ± 0.14 and 3.5 ± 0.4 ms with a predominant conductance of 26 pS. α3β4 AChRs had open times of 1.4 ± 0.2 and 6.5 ± 0.8 ms and a predominant conductance of 31 pS. Burst times were 0.82 ± 0.12 and 5.3 ± 0.7 ms for α3β2 and 1.7 ± 0.1 and 16 ± 1 ms for α3β4. Desensitization was faster for AChRs with the β2 subunit than for those with the β4 subunit.One open time for α3α5β2 AChRs (5.5 ± 0.3 ms) was different from those of α3β2 AChRs. For α3α5β4 AChRs, an additional conductance, open time and burst time (36 pS, 22 ± 3 ms and 43 ± 4 ms, respectively) were different from those for α3β4 AChRs.α3 AChRs were inhibited by hexamethonium or mecamylamine. The rate constants for block of α3β4 by hexamethonium and of α3β2 by mecamylamine were 1.2 × 107 and 4.6 × 107 M−1 s−1, respectively.AChRs from IMR-32 cells had a predominant conductance of 32 pS and open times of 1.5 ± 0.3 and 9.6 ± 1.2 ms. These properties were most similar to those of α3β4 AChRs expressed in oocytes. Antibodies revealed that 5 ± 2% of IMR-32 α3 AChRs contained α5 subunits and 6 ± 2% contained β2 subunits. IMR-32 α3 AChRs are primarily α3β4 AChRs. PMID:10200416

Galactose alters markers of oxidative stress and acetylcholinesterase activity in the cerebrum of rats: protective role of antioxidants.

PubMed

Delwing-de Lima, Daniela; Fröhlich, Monique; Dalmedico, Leticia; Aurélio, Juliana Gruenwaldt Maia; Delwing-Dal Magro, Débora; Pereira, Eduardo Manoel; Wyse, Angela T S

2017-04-01

We evaluated the in vitro effects of galactose at 0.1, 3.0, 5.0 and 10.0 mM on thiobarbituric acid-reactive substances (TBA-RS), total sulfhydryl content, protein carbonyl content, on the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and on acetylcholinesterase (AChE) activity in the cerebral cortex, cerebellum and hippocampus of rats. We also investigated the influence of the antioxidants (each at 1 mM), α-tocopherol, ascorbic acid and glutathione, on the effects elicited by galactose on the parameters tested. Results showed that galactose, at a concentration of 3.0 mM, enhanced TBA-RS levels in the hippocampus, cerebral cortex and cerebellum of rats. In the cerebral cortex, galactose at concentrations of 5.0 and 10.0 mM increased TBA-RS and protein carbonyl content, and at 10.0 mM increased CAT activity and decreased AChE activity. In the cerebellum, galactose at concentrations of 5.0 and 10.0 mM increased TBA-RS, SOD and GSH-Px activities. In the hippocampus, galactose at concentrations of 5.0 and 10.0 mM increased TBA-RS and CAT activity and at 10.0 mM decreased GSH-Px. Data showed that at the pathologically high concentration (greater than 5.0 mM), galactose induces lipid peroxidation, protein carbonylation, alters antioxidant defenses in the cerebrum, and also alters cholinesterase activity. Trolox, ascorbic acid and glutathione addition prevented the majority of alterations in oxidative stress parameters and the decrease in AChE activity that were caused by galactose. Our findings lend support to a potential therapeutic strategy for this condition, which may include the use of appropriate antioxidants for ameliorating the damage caused by galactose.

Nicotine suppresses the neurotoxicity by MPP+/MPTP through activating α7nAChR/PI3K/Trx-1 and suppressing ER stress.

PubMed

Cai, Yanxue; Zhang, Xianwen; Zhou, Xiaoshuang; Wu, Xiaoli; Li, Yanhui; Yao, Jianhua; Bai, Jie

2017-03-01

Parkinson's disease (PD) is a neurodegenerative disease. Nicotine has been reported to have the role in preventing Parkinson's disease. However, its mechanism is still unclear. In present study we found that nicotine suppressed 1-methyl-4-phenylpyridinium ion(MPP + ) toxicity in PC12 cells by MTT assay. The expression of thioredoxin-1(Trx-1) was decreased by MPP + , which was restored by nicotine. The nicotine suppressed expressions of Glucose-regulated protein 78(GRP78/Bip) and C/EBP homologous protein (CHOP) induced by MPP + . The methyllycaconitine (MLA), the inhibitor of α7nAChR and LY294002, the inhibitor of phosphatidylinositol 3-kinase (PI3K) blocked the suppressions of above molecules, respectively. Consistently, pretreatment with nicotine ameliorated the motor ability, restored the declines of Trx-1 and tyrosine hydroxylase (TH), and suppressed the expressions of Bip and CHOP induced by 1-Methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Our results suggest that nicotine plays role in resisting MPP + /MPTP neurotoxicity through activating the α7nAChR/PI3K/Trx-1 pathway and suppressing ER stress. Copyright © 2017 Elsevier B.V. All rights reserved.

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. Copyright © 2016. Published by Elsevier Ireland Ltd.

Mouse auditory cortex differs from visual and somatosensory cortices in the laminar distribution of cytochrome oxidase and acetylcholinesterase.

PubMed

Anderson, L A; Christianson, G B; Linden, J F

2009-02-03

Cytochrome oxidase (CYO) and acetylcholinesterase (AChE) staining density varies across the cortical layers in many sensory areas. The laminar variations likely reflect differences between the layers in levels of metabolic activity and cholinergic modulation. The question of whether these laminar variations differ between primary sensory cortices has never been systematically addressed in the same set of animals, since most studies of sensory cortex focus on a single sensory modality. Here, we compared the laminar distribution of CYO and AChE activity in the primary auditory, visual, and somatosensory cortices of the mouse, using Nissl-stained sections to define laminar boundaries. Interestingly, for both CYO and AChE, laminar patterns of enzyme activity were similar in the visual and somatosensory cortices, but differed in the auditory cortex. In the visual and somatosensory areas, staining densities for both enzymes were highest in layers III/IV or IV and in lower layer V. In the auditory cortex, CYO activity showed a reliable peak only at the layer III/IV border, while AChE distribution was relatively homogeneous across layers. These results suggest that laminar patterns of metabolic activity and cholinergic influence are similar in the mouse visual and somatosensory cortices, but differ in the auditory cortex.

Effects of dietary lead acetate on hepatic detoxication enzyme activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagstaff, D.J.

1979-12-01

Lead-containing compounds usually inhibit enzymic and metabolic processes. This inhibition is presumed to be the mechanism of intoxication by these compounds. Inhibition of detoxication activities of liver microsomal enzymes could be particularly detrimental because the toxicity of many different substances would be increased. Exposure of experimental animals to lead compounds in several studies has been associated with depressed activity of hepatic microsomal enzymes, reduced levels of hepatic cytochrome P-450, reduced levels of hepatic microsomal protein, and prolonged hexobarbital sleep times. The present report contains observations that under certain experimental conditions there is stimulated hepatic meicrosomal enzyme activity in rats fedmore » lead acetate.« less

Optical detection of organophosphorus compounds based on Mn-doped ZnSe d-dot enzymatic catalytic sensor.

PubMed

Gao, Xue; Tang, Guangchao; Su, Xingguang

2012-01-01

In this paper, we report a sensitive and selective method for detection of organophosphorus compounds (OPs) based on Mn:ZnSe d-dots-enzyme-hydrogen peroxide (H(2)O(2)) fluorescence quenching system. Acetylcholine esterase (AChE) can hydrolyze acetylcholine (ACh) to choline. Subsequently, choline oxidase (ChOx) oxidizes choline to generate H(2)O(2). The enzyme-generated H(2)O(2) can quench the fluorescence of Mn:ZnSe d-dots. When paraoxon are introduced in solution, it can interact with the active centers of AChE and decrease the enzyme activity. This leads to the decrease of the H(2)O(2) production and then the fluorescence quenching rate of Mn:ZnSe d-dots. Experimental results showed that the enzyme inhibition percentage of Mn:ZnSe d-dots-ChOx-AChE-ACh system was proportional to the logarithm of paraoxon in the range 4.84×10(-11) to 4.84×10(-6) mol/L with the detection limit (S/N=3) of 1.31×10(-11) mol/L. The proposed biosensor has been employed for quick determination of paraoxon in tap water and milk samples with satisfactory reproducibility and accuracy. This nano-biosensor was proved to be sensitive, rapid, simple and tolerance of most interfering substances. Copyright © 2012 Elsevier B.V. All rights reserved.

Discriminative structural approaches for enzyme active-site prediction.

PubMed

Kato, Tsuyoshi; Nagano, Nozomi

2011-02-15

Predicting enzyme active-sites in proteins is an important issue not only for protein sciences but also for a variety of practical applications such as drug design. Because enzyme reaction mechanisms are based on the local structures of enzyme active-sites, various template-based methods that compare local structures in proteins have been developed to date. In comparing such local sites, a simple measurement, RMSD, has been used so far. This paper introduces new machine learning algorithms that refine the similarity/deviation for comparison of local structures. The similarity/deviation is applied to two types of applications, single template analysis and multiple template analysis. In the single template analysis, a single template is used as a query to search proteins for active sites, whereas a protein structure is examined as a query to discover the possible active-sites using a set of templates in the multiple template analysis. This paper experimentally illustrates that the machine learning algorithms effectively improve the similarity/deviation measurements for both the analyses.

Enhanced synthesis and release of dopamine in transgenic mice with gain-of-function α6* nAChRs.

PubMed

Wang, Yuexiang; Lee, Jang-Won; Oh, Gyeon; Grady, Sharon R; McIntosh, J Michael; Brunzell, Darlene H; Cannon, Jason R; Drenan, Ryan M

2014-04-01

α6β2* nicotinic acetylcholine receptors (nAChRs)s in the ventral tegmental area to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6β2* nAChRs (α6L9'S mice) that are hypersensitive to nicotine and endogenous acetylcholine. Evoked extracellular dopamine (DA) levels were enhanced in α6L9'S NAc slices compared to control, non-transgenic (non-Tg) slices. Extracellular DA levels in both non-Tg and α6L9'S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by acetylcholine plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9'S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9'S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid compared to non-Tg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid, and norepinephrine (NE) were unchanged in α6L9'S compared to non-Tg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9'S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels. © 2013 International Society for Neurochemistry.

Synthesis of some new 3-coumaranone and coumarin derivatives as dual inhibitors of acetyl- and butyrylcholinesterase.

PubMed

Alipour, Masoumeh; Khoobi, Mehdi; Nadri, Hamid; Sakhteman, Amirhossein; Moradi, Alireza; Ghandi, Mehdi; Foroumadi, Alireza; Shafiee, Abbas

2013-08-01

A novel series of coumarin and 3-coumaranone derivatives encompassing the phenacyl pyridinium moiety were synthesized and evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity using Ellman's method. All compounds presented inhibitory activity against both AChE and BuChE in the micromolar range. The molecular docking simulations revealed that all compounds were dual binding site inhibitors of AChE. A kinetic study was performed and the mechanism of enzyme inhibition was proved to be of mixed type. All compounds were tested for their antioxidant activity and no significant activity was observed. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Patterns of functional enzyme activity in fungus farming ambrosia beetles.

PubMed

De Fine Licht, Henrik H; Biedermann, Peter H W

2012-06-06

In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray

A Simple and Accurate Method for Measuring Enzyme Activity.

ERIC Educational Resources Information Center

Yip, Din-Yan

1997-01-01

Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

Application of activity-based protein profiling to study enzyme function in adipocytes.

PubMed

Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F; Saez, Enrique

2014-01-01

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. © 2014 Elsevier Inc. All rights reserved.

Molecular architectures and functions of radical enzymes and their (re)activating proteins.

PubMed

Shibata, Naoki; Toraya, Tetsuo

2015-10-01

Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes—i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Evolutionary transitions in enzyme activity of ant fungus gardens.

PubMed

De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

2010-07-01

Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications

PubMed Central

Ren, Lili; Chen, Fang; Feng, Yuqian

2016-01-01

Ginkgo biloba is a typical relic plant that rarely suffers from pest hazards. This study analyzed the pattern of G. biloba pest hazards in Beijing; tested the antifeedant activity of G. biloba extracts, including ginkgo flavonoids, ginkgolide, and bilobalide, against Hyphantria cunea larvae; determined the activities of glutathione transferase (GSTs), acetylcholinesterase (AChE), carboxylesterase (CarE) and mixed-functional oxidase (MFO), in larvae after feeding on these G. biloba secondary metabolites; and screened for effective botanical antifeedants in the field. In this study, no indicators of insect infestation were found for any of the examined leaves of G. biloba; all tested secondary metabolites showed significant antifeedant activity and affected the activity of the four larval detoxifying enzymes. Ginkgolide had the highest antifeedant activity and the most significant effect on the detoxifying enzymes (P<0.05). Spraying leaves with G. biloba extracts or ginkgolide both significantly repelled H. cunea larvae in the field (P<0.05), although the former is more economical and practical. This study investigated the antifeedant activity of G. biloba secondary metabolites against H. cunea larvae, and the results provide new insights into the mechanism of G. biloba pest resistance. This study also developed new applications of G. biloba secondary metabolites for effective pest control. PMID:27214257

Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications.

PubMed

Pan, Long; Ren, Lili; Chen, Fang; Feng, Yuqian; Luo, Youqing

2016-01-01

Ginkgo biloba is a typical relic plant that rarely suffers from pest hazards. This study analyzed the pattern of G. biloba pest hazards in Beijing; tested the antifeedant activity of G. biloba extracts, including ginkgo flavonoids, ginkgolide, and bilobalide, against Hyphantria cunea larvae; determined the activities of glutathione transferase (GSTs), acetylcholinesterase (AChE), carboxylesterase (CarE) and mixed-functional oxidase (MFO), in larvae after feeding on these G. biloba secondary metabolites; and screened for effective botanical antifeedants in the field. In this study, no indicators of insect infestation were found for any of the examined leaves of G. biloba; all tested secondary metabolites showed significant antifeedant activity and affected the activity of the four larval detoxifying enzymes. Ginkgolide had the highest antifeedant activity and the most significant effect on the detoxifying enzymes (P<0.05). Spraying leaves with G. biloba extracts or ginkgolide both significantly repelled H. cunea larvae in the field (P<0.05), although the former is more economical and practical. This study investigated the antifeedant activity of G. biloba secondary metabolites against H. cunea larvae, and the results provide new insights into the mechanism of G. biloba pest resistance. This study also developed new applications of G. biloba secondary metabolites for effective pest control.

Nicergoline reverts haloperidol-induced loss of detoxifying-enzyme activity.

PubMed

Vairetti, Mariapia; Ferrigno, Andrea; Canonico, Pier Luigi; Battaglia, Angelo; Bertè, Francantonio; Richelmi, Plinio

2004-11-28

We evaluated the effects of nicergoline on antioxidant defense enzymes (detoxifying enzymes), during chronic treatment with haloperidol in rats. Chronic use of haloperidol (10 weeks, 1.5 mg/kg/day) induces a significant decrease in glutathione reductase, glutathione peroxidase and superoxide dismutase activity, in selected areas of the brain. Co-administration of nicergoline (20 days, 10 mg/kg/day) significantly restored the activity of these enzymes to levels comparable to those observed in control rats. These observations suggest beneficial effects of nicergoline in the prevention and in the treatment of haloperidol-induced side effects.

Novel approaches to study the involvement of α7-nAChR in human diseases.

PubMed

Palma, Eleonora; Conti, Luca; Roseti, Cristina; Limatola, Cristina

2012-05-01

The alpha7 nicotinic acetylcholine receptor (α7 nAChR) is widely distributed in the human brain and has been implicated in a number of human central nervous system (CNS) diseases, including Alzheimer's and Parkinson's disease, schizophrenia and autism. Recently, new roles for α7 nAChRs in lung cancer and heart disease have been elucidated. Despite the importance of this receptor in human pathology, many technical difficulties are still encountered when investigating the role of α7 nAChRs. Electrophysiological analysis of the receptor upon heterologous expression or in human tissues was limited by the fast desensitization of α7-mediated nicotinic currents and by tissue availability. In addition, animal models for the human diseases related to α7 nAChRs have long been unavailable. The recent development of new imaging and analysis approaches such as PET and receptor microtransplantation have rendered the study of α7 nAChRs increasingly feasible, paving new roads to the design of therapeutic drugs. This review summarizes the current knowledge and recent findings obtained by these novel approaches.

Current concepts on selected plant secondary metabolites with promising inhibitory effects against enzymes linked to Alzheimer's disease.

PubMed

Orhan, I Erdogan

2012-01-01

Alzheimer's disease (AD) has become one of the deadliest diseases for human beings with special incidence in elderly population. It is a progressive neurodegenerative disease and the most prevalent cause of dementia. The neuropathology of AD has not been fully elucidated yet, however, cholinergic hypothesis is the most accepted theory nowadays, resulting from the cholinergic deficit emerging in the brains of AD patients. Shortage of the neurotransmitters, acetylcholine and butyrylcholine has been demonstrated, and therefore, inhibition of the enzymes; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that break down acetylcholine and butyrylcholine has become a standard approach for AD treatment. However, cholinesterase inhibitors are only effective in symptomatic treatment and have no ability to impede the disease. The pathogenesis of AD is highly complex and another hypothesis is the formation of amyloid plaques containing beta-amyloid peptide, which causes neurolesions in the brains of AD patients. Beta-amyloid peptide is generated after the sequential cleavage of amyloid precursor protein, especially by the beta- and gamma-secretase in the amyloidogenic pathway. The secretases involved in the processing of amyloid precursor protein are of particular interest and, consequently, the inhibition of secretase enzyme family of protease type has become another desired treatment strategy for AD. On the other hand, medicinal plants are attractive sources for drug research and development as they produce chemically-varying molecules with preferred biological activities. The aim of this article is to review the available data on selected inhibitors from plant secondary metabolites with emphasis on cholinesterase, prolyl endopeptidase, and secretase enzyme families as being the current treatments of AD.

Effects of epithalon on activities gastrointestinal enzymes in young and old rats.

PubMed

Khavinson, V Kh; Malinin, V V; Timofeeva, N M; Egorova, V V; Nikitina, A A

2002-03-01

Peroral administration of Epithalon (Ala-Glu-Asp-Gly) to male and female Wistar rats aging 3 and 11 months changed activity of enzymes hydrolyzing carbohydrates, proteins, and phosphoric acid esters in various portions of the gastrointestinal tract. The most pronounced activation of enzymes was observed in 11-month-old animals. This effect diminished the differences in enzyme activities between young and old rats (compared to untreated animals). Our results indicate that Epithalon modulates activity of gastrointestinal enzymes during aging.

Defying the activity-stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability.

PubMed

Siddiqui, Khawar Sohail

2017-05-01

The biotechnological applications of enzymes are limited due to the activity-stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity-stability trade-off can be overcome. In this review, the origin of the activity-stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity-stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel-Danson equilibrium model, catalytic antibodies, and life on cold planets.

Blockade of Neuronal α7-nAChR by α-Conotoxin ImI Explained by Computational Scanning and Energy Calculations

PubMed Central

Yu, Rilei; Craik, David J.; Kaas, Quentin

2011-01-01

α-Conotoxins potently inhibit isoforms of nicotinic acetylcholine receptors (nAChRs), which are essential for neuronal and neuromuscular transmission. They are also used as neurochemical tools to study nAChR physiology and are being evaluated as drug leads to treat various neuronal disorders. A number of experimental studies have been performed to investigate the structure-activity relationships of conotoxin/nAChR complexes. However, the structural determinants of their binding interactions are still ambiguous in the absence of experimental structures of conotoxin-receptor complexes. In this study, the binding modes of α-conotoxin ImI to the α7-nAChR, currently the best-studied system experimentally, were investigated using comparative modeling and molecular dynamics simulations. The structures of more than 30 single point mutants of either the conotoxin or the receptor were modeled and analyzed. The models were used to explain qualitatively the change of affinities measured experimentally, including some nAChR positions located outside the binding site. Mutational energies were calculated using different methods that combine a conformational refinement procedure (minimization with a distance dependent dielectric constant or explicit water, or molecular dynamics using five restraint strategies) and a binding energy function (MM-GB/SA or MM-PB/SA). The protocol using explicit water energy minimization and MM-GB/SA gave the best correlations with experimental binding affinities, with an R2 value of 0.74. The van der Waals and non-polar desolvation components were found to be the main driving force for binding of the conotoxin to the nAChR. The electrostatic component was responsible for the selectivity of the various ImI mutants. Overall, this study provides novel insights into the binding mechanism of α-conotoxins to nAChRs and the methodological developments reported here open avenues for computational scanning studies of a rapidly expanding range of wild

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.

PubMed

Santiago, Margarita; Ramírez-Sarmiento, César A; Zamora, Ricardo A; Parra, Loreto P

2016-01-01

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

Brain acetycholinesterase activity in botulism-intoxicated mallards

USGS Publications Warehouse

Rocke, T.E.; Samuel, M.D.

1991-01-01

Brain acetylcholinesterase (AChE) activity in captive-reared mallards (Anas platyrhynchos) that died of botulism was compared with euthanized controls. AChE levels for both groups were within the range reported for normal mallards, and there was no significant difference in mean AChE activity between birds that ingested botulism toxin and died and those that did not.

Synthesis, characterization, DNA-Binding, enzyme inhibition and antioxidant studies of new N-methylated derivatives of pyridinium amine

NASA Astrophysics Data System (ADS)

Zafar, Muhammad Naveed; Perveen, Fouzia; Nazar, Muhammad Faizan; Mughal, Ehsan Ullah; Rafique, Humera; Tahir, Muhammad Nawaz; Akbar, Muhammad Sharif; Zahra, Sabeen

2017-06-01

A series of novel N-methylated derivatives of pyridinium amine, [L1][Tf]-[L5][Tf], were synthesized and characterized by FTIR, NMR, MS and XRD analyses. Preliminary biological screening of these compounds including antioxidant, enzyme inhibition and DNA (salmon sperm) interaction studies were also carried out. The targeted compounds were synthesized by a melt reaction between 4-chloro-N-methyl pyridinium triflate and corresponding amines (1-naphthyl amine, o-ansidine, 2-nitroaniline, p-ansidine and cyclohexyl amine) at temperature of 230 °C. The DPPH radical antioxidant scavenging activities of these compounds at maximum concentration of 50 μg/mL were observed in the range of 60-70%. Acetylcholine esterase (AChE) and Butylcholine esterase (BChE) inhibitory activities of synthesized compounds at 2 mM concentration were also measured to be at maximum of 79 and 71% respectively. The spectral behavior of ligand-DNA obtained from photo-luminescent measurements showed that all ligands bind with DNA via non-covalent interactions. The binding constant values were determined by UV-visible and fluorescence spectroscopy and were quite close to that obtained from molecular docking studies.

Synthesis and cholinesterase inhibition of cativic acid derivatives.

PubMed

Alza, Natalia P; Richmond, Victoria; Baier, Carlos J; Freire, Eleonora; Baggio, Ricardo; Murray, Ana Paula

2014-08-01

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. In a preliminary study, significant AChE inhibition was observed for the ethanolic extract of Grindelia ventanensis (IC₅₀=0.79 mg/mL). This result prompted us to isolate the active constituent, a normal labdane diterpenoid identified as 17-hydroxycativic acid (1), through a bioassay guided fractionation. Taking into account that 1 showed moderate inhibition of AChE (IC₅₀=21.1 μM), selectivity over butyrylcholinesterase (BChE) (IC₅₀=171.1 μM) and that it was easily obtained from the plant extract in a very good yield (0.15% w/w), we decided to prepare semisynthetic derivatives of this natural diterpenoid through simple structural modifications. A set of twenty new cativic acid derivatives (3-6) was prepared from 1 through transformations on the carboxylic group at C-15, introducing a C2-C6 linker and a tertiary amine group. They were tested for their inhibitory activity against AChE and BChE and some structure-activity relationships were outlined. The most active derivative was compound 3c, with an IC₅₀ value of 3.2 μM for AChE. Enzyme kinetic studies and docking modeling revealed that this inhibitor targeted both the catalytic active site and the peripheral anionic site of this enzyme. Furthermore, 3c showed significant inhibition of AChE activity in SH-SY5Y human neuroblastoma cells, and was non-cytotoxic. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Effects of Exercise on Pharmacokinetics and Pharmacodynamics of Physostigmine in Rats

DTIC Science & Technology

1991-02-15

combination of the two, regulate the biosynthetic and degradative enzymes of ACh in EDL (extensor digitorum longus) - fast muscle and soleus - slow muscle...and trained exercise on ChAT activities in fast and slow muscles of rat 100 17. Effect of subacute Phy (70 4g/kg, i.m.) and endurance training on...AChE activities in EDL and soleus muscles of rat 100 18. ChAT and AChE activities (% of control) in fast (EDL) and slow (soleus) muscle in subacute Phy

Temperature and UV light affect the activity of marine cell-free enzymes

NASA Astrophysics Data System (ADS)

Thomson, Blair; Hepburn, Christopher David; Lamare, Miles; Baltar, Federico

2017-09-01

Microbial extracellular enzymatic activity (EEA) is the rate-limiting step in the degradation of organic matter in the oceans. These extracellular enzymes exist in two forms: cell-bound, which are attached to the microbial cell wall, and cell-free, which are completely free of the cell. Contrary to previous understanding, cell-free extracellular enzymes make up a substantial proportion of the total marine EEA. Little is known about these abundant cell-free enzymes, including what factors control their activity once they are away from their sites (cells). Experiments were run to assess how cell-free enzymes (excluding microbes) respond to ultraviolet radiation (UVR) and temperature manipulations, previously suggested as potential control factors for these enzymes. The experiments were done with New Zealand coastal waters and the enzymes studied were alkaline phosphatase (APase), β-glucosidase, (BGase), and leucine aminopeptidase (LAPase). Environmentally relevant UVR (i.e. in situ UVR levels measured at our site) reduced cell-free enzyme activities by up to 87 % when compared to controls, likely a consequence of photodegradation. This effect of UVR on cell-free enzymes differed depending on the UVR fraction. Ambient levels of UV radiation were shown to reduce the activity of cell-free enzymes for the first time. Elevated temperatures (15 °C) increased the activity of cell-free enzymes by up to 53 % when compared to controls (10 °C), likely by enhancing the catalytic activity of the enzymes. Our results suggest the importance of both UVR and temperature as control mechanisms for cell-free enzymes. Given the projected warming ocean environment and the variable UVR light regime, it is possible that there could be major changes in the cell-free EEA and in the enzymes contribution to organic matter remineralization in the future.

A review on the effects of supercritical carbon dioxide on enzyme activity.

PubMed

Wimmer, Zdenek; Zarevúcka, Marie

2010-01-19

Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO(2). The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability.

A Review on the Effects of Supercritical Carbon Dioxide on Enzyme Activity

PubMed Central

Wimmer, Zdeněk; Zarevúcka, Marie

2010-01-01

Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO2. The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability. PMID:20162013

Evaluation of fruit extracts of six Turkish Juniperus species for their antioxidant, anticholinesterase and antimicrobial activities.

PubMed

Oztürk, Mehmet; Tümen, İbrahim; Uğur, Aysel; Aydoğmuş-Öztürk, Fatma; Topçu, Gülaçtı

2011-03-30

Juniperus L. (Cupressaceae) species are mostly spread out in the Northern Hemisphere of the world, and some of them are used as folkloric medicines. The fruits of some species are eaten. Since oxidative stress is one of the reasons for neurodegeneration and is associated with the Alzheimer's disease (AD), the extracts prepared from the fruits of six Juniperus species were screened for their antioxidant activity. Therefore, the extracts were also evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which are chief enzymes in the pathogenesis of AD. In addition, antimicrobial activity was also evaluated. In the β-carotene-linoleic acid assay, acetone extracts of J. oxycedrus subsp. oxycedrus, J. sabina and J. excelsa, and methanol extracts of J. phoenicea and J. sabina, effectively inhibited oxidation of linoleic acid. The hexane extracts of J. oxycedrus subsp. oxycedrus, J. foetidissima and J. phoenicea showed remarkable inhibitory effect against AChE and BChE. Because of their high antioxidant activity, J. excelsa, J. oxycedrus subsp. oxycedrus, J. sabina and J. phoenicia might be used in the food industry as preservative agents or extension of the shelf-life of raw and processed foods. Since the hexane extracts of J. oxycedrus subsp. oxycedrus and J. foetidissima demonstrated significant anticholinesterase activity they should be considered as a potential source for anticholinesterase agents. Copyright © 2010 Society of Chemical Industry.

SN56 neuronal cell death after 24 h and 14 days chlorpyrifos exposure through glutamate transmission dysfunction, increase of GSK-3β enzyme, β-amyloid and tau protein levels.

PubMed

Moyano, Paula; Frejo, María Teresa; Anadon, María José; García, José Manuel; Díaz, María Jesús; Lobo, Margarita; Sola, Emma; García, Jimena; Del Pino, Javier

2018-06-01

Chlorpyrifos (CPF) is an organophosphate insecticide described to induce cognitive disorders, both after acute and repeated administration. However, the mechanisms through which it induces these effects are unknown. CPF has been reported to produce basal forebrain cholinergic neuronal cell death, involved on learning and memory regulation, which could be the cause of such cognitive disorders. Neuronal cell death was partially mediated by oxidative stress generation, P75 NTR and α 7 -nAChRs gene expression alteration triggered through acetylcholinesterase (AChE) variants disruption, suggesting other mechanisms are involved. In this regard, CPF induces Aβ and tau proteins production and activation of GSK3β enzyme and alters glutamatergic transmission, which have been related with basal forebrain cholinergic neuronal cell death and development of cognitive disorders. According to these data, we hypothesized that CPF induces basal forebrain cholinergic neuronal cell death through induction of Aβ and tau proteins production, activation of GSK-3β enzyme and disruption of glutamatergic transmission. We evaluated this hypothesis in septal SN56 basal forebrain cholinergic neurons, after 24 h and 14 days CPF exposure. This study shows that CPF increases glutamate levels, upregulates GSK-3β gene expression, and increases the production of Aβ and phosphorylated tau proteins and all these effects reduced cell viability. CPF increases glutaminase activity and upregulates the VGLUT1 gene expression, which could mediate the disruption of glutamatergic transmission. Our present results provide new understanding of the mechanisms contributing to the harmful effects of CPF, and its possible relevance in the pathogenesis of neurodegenerative diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Enzyme activities in plasma, kidney, liver, and muscle of five avian species

USGS Publications Warehouse

Franson, J.C.; Murray, H.C.; Bunck, C.

1985-01-01

Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH) were determined in plasma, kidney, liver, and muscle from five species of captive birds. Few differences occurred in plasma activities between sexes but considerable differences occurred between species. All five enzymes were detected in each of the tissues sampled. Relative enzyme activities in liver, kidney, and muscle were similar for each species. CPK activity was much higher in muscle than in liver or kidney and, of the five enzymes studied, may be the best indicator of muscle damage. Most of the other enzymes were more evenly distributed among the three tissues, and no organ-specific enzyme could be identified for liver or kidney. Because of interspecific variations in plasma enzyme activities, it is important to establish baseline values for each species to ensure accurate interpretation of results.

Toxicological and biochemical basis of synergism between the entomopathogenic fungus Lecanicillium muscarium and the insecticide matrine against Bemisia tabaci (Gennadius)

NASA Astrophysics Data System (ADS)

Ali, Shaukat; Zhang, Can; Wang, Zeqing; Wang, Xing-Min; Wu, Jian-Hui; Cuthbertson, Andrew G. S.; Shao, Zhenfang; Qiu, Bao-Li

2017-04-01

The sweetpotato whitefly Bemisia tabaci (Gennadius) was challenged with different combinations of matrine (insecticide) and Lecanicillium muscarium (entomopathogenic fungus). Our results revealed a synergistic relationship between matrine and L. muscarium on mortality and enzyme activities of B. tabaci. To illustrate the biochemical mechanisms involved in detoxification and immune responses of B. tabaci against both control agents, activities of different detoxifying and antioxidant enzymes were quantified. After combined application of matrine and L. muscarium, activities of carboxylestrease (CarE), glutathione-s-transferase (GSTs) and chitinase (CHI) decreased during the initial infection period. Acetylcholinestrase (AChE) activities increased during the entire experimental period, whereas those of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) decreased during the later infection period. The increased mortality and suppression of enzymatic response of B. tabaci following matrine and L. muscarium application suggests a strong synergistic effect between both agents. The strong synergistic effect is possibly related to the disturbance of acetylcholine balance and changes in AchE activities of the whitefly as both matrine and L. muscarium target insect acetylcholine (Ach) receptors which in turn effects AchE production. Therefore, our results have revealed the complex biochemical processes involved in the synergistic action of matrine and L. muscarium against B. tabaci.

Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

NASA Astrophysics Data System (ADS)

Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2015-04-01

Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil

Development of M1 mAChR allosteric and bitopic ligands: prospective therapeutics for the treatment of cognitive deficits.

PubMed

Davie, Briana J; Christopoulos, Arthur; Scammells, Peter J

2013-07-17

Since the cholinergic hypothesis of memory dysfunction was first reported, extensive research efforts have focused on elucidating the mechanisms by which this intricate system contributes to the regulation of processes such as learning, memory, and higher executive function. Several cholinergic therapeutic targets for the treatment of cognitive deficits, psychotic symptoms, and the underlying pathophysiology of neurodegenerative disorders, such as Alzheimer's disease and schizophrenia, have since emerged. Clinically approved drugs now exist for some of these targets; however, they all may be considered suboptimal therapeutics in that they produce undesirable off-target activity leading to side effects, fail to address the wide variety of symptoms and underlying pathophysiology that characterize these disorders, and/or afford little to no therapeutic effect in subsets of patient populations. A promising target for which there are presently no approved therapies is the M1 muscarinic acetylcholine receptor (M1 mAChR). Despite avid investigation, development of agents that selectively activate this receptor via the orthosteric site has been hampered by the high sequence homology of the binding site between the five muscarinic receptor subtypes and the wide distribution of this receptor family in both the central nervous system (CNS) and the periphery. Hence, a plethora of ligands targeting less structurally conserved allosteric sites of the M1 mAChR have been investigated. This Review aims to explain the rationale behind allosterically targeting the M1 mAChR, comprehensively summarize and critically evaluate the M1 mAChR allosteric ligand literature to date, highlight the challenges inherent in allosteric ligand investigation that are impeding their clinical advancement, and discuss potential methods for resolving these issues.

De novo active sites for resurrected Precambrian enzymes

NASA Astrophysics Data System (ADS)

Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

2017-07-01

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

Enzyme activation through the utilization of intrinsic dianion binding energy.

PubMed

Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

2017-03-01

We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

PubMed

Li, Guang-Qi; Zang, Xiao-Nan; Zhang, Xue-Cheng; Lu, Ning; Ding, Yan; Gong, Le; Chen, Wen-Chao

2014-03-15

To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

PubMed

Yuan, Lin; Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

PubMed Central

Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

NASA Astrophysics Data System (ADS)

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-02-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

PubMed Central

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-01-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

PubMed

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

2016-02-10

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

AT–1001: a high-affinity α3β4 nAChR ligand with novel nicotine-suppressive pharmacology

PubMed Central

Cippitelli, Andrea; Wu, Jinhua; Gaiolini, Kelly A; Mercatelli, Daniela; Schoch, Jennifer; Gorman, Michelle; Ramirez, Alejandra; Ciccocioppo, Roberto; Khroyan, Taline V; Yasuda, Dennis; Zaveri, Nurulain T; Pascual, Conrado; Xie, Xinmin (Simon); Toll, Lawrence

2015-01-01

Background and Purpose The α3β4 subtype of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating nicotine reinforcement processes. AT-1001 has been recently described as a high-affinity and selective α3β4 nAChR antagonist that blocks nicotine self-administration in rats. The aim of this study was to investigate the mechanism of action underlying the nicotine-suppressive effects of AT-1001. Experimental Approach Effects of AT-1001 were determined using in vitro assays and rat models of nicotine addiction, and compared with varenicline. Key Results AT-1001 and its analogue AT-1012 were functionally selective as antagonists for α3β4 over α4β2 nAChRs, but not to the same extent as the binding selectivity, and had partial agonist activity at α3β4 nAChRs. In contrast, varenicline was a partial agonist at α4β2, a weak agonist at α3β4 and inhibited α4β2 at a much lower concentration than it inhibited α3β4 nAChRs. AT-1001 and varenicline also had very different in vivo properties. Firstly, AT-1001 did not exhibit reinforcing properties per se while varenicline was self-administered. Secondly, systemic treatment with AT-1001 did not induce reinstatement of nicotine seeking but rather attenuated reinstatement induced by varenicline, as well as nicotine. Finally, unlike varenicline, AT-1001 selectively blocked nicotine self-administration without altering alcohol lever pressing as assessed in an operant co-administration paradigm. Conclusions and Implications These findings describe a more complex AT-1001 in vitro profile than previously appreciated and provide further support for the potential of AT-1001 and congeners as clinically useful compounds for smoking cessation, with a mechanism of action distinct from currently available medications. PMID:25440006

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.

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

PubMed Central

Santiago, Margarita; Ramírez-Sarmiento, César A.; Zamora, Ricardo A.; Parra, Loreto P.

2016-01-01

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications. PMID:27667987

Aqueous extracts of avocado pear (Persea americana Mill.) leaves and seeds exhibit anti-cholinesterases and antioxidant activities in vitro.

PubMed

Oboh, Ganiyu; Odubanjo, Veronica O; Bello, Fatai; Ademosun, Ayokunle O; Oyeleye, Sunday I; Nwanna, Emem E; Ademiluyi, Adedayo O

2016-03-01

Avocado pear (Persea americana Mill.) leaves and seeds are used in traditional medicine for the treatment/management of Alzheimer disease (AD); however, information on the mechanism of actions is limited. This study sought to investigate the effect of P. americana leaf and seed aqueous extracts on some enzymes linked with AD (acetylcholinesterase [AChE] and butyrylcholinesterase [BChE] activities) and their antioxidant potentials in vitro. The inhibitory effects of extracts on AChE and BChE activities and antioxidant potentials (inhibition of Fe2+- and sodium nitroprusside-induced thiobarbiturate reactive species [TBARS] production in rat brain homogenates, radicals [1,1-diphenyl-2-picrylhydrazyl, hydroxyl, and nitric oxide] scavenging and iron [Fe] chelation abilities) were investigated. Phenolic content and phytochemical screening were carried out. Alkaloid profile was also determined using gas chromatography coupled with flame ionization detector (GC-FID). The extracts inhibited AChE and BChE activities and prooxidant-induced TBARS production in a dose-dependent manner, with the seed extract having the highest inhibitory effect and the leaf extract exhibiting higher phenolic content and radical scavenging abilities, but lower Fe chelation ability compared with that of the seed. The phytochemical screening revealed the presence of saponins, alkaloids, and terpenoids in both extracts, whereas the total alkaloid profile was higher in the seed extract than in the leaf extract, as revealed by GC-FID. The anti-cholinesterase and antioxidant activities of avocado leaf and seed could be linked to their phytoconstituents and might be the possible mechanisms underlying their use as a cheap and natural treatment/management of AD. However, these extracts should be further investigated in vivo.

Effects of Different Substrates on Lignocellulosic Enzyme Expression, Enzyme Activity, Substrate Utilization and Biological Efficiency of Pleurotus Eryngii.

PubMed

Xie, Chunliang; Yan, Li; Gong, Wenbing; Zhu, Zuohua; Tan, Senwei; Chen, Du; Hu, Zhenxiu; Peng, Yuande

2016-01-01

Pleurotus eryngii is one of the most valued and delicious mushrooms which are commercially cultivated on various agro-wastes. How different substrates affect lignocellulosic biomass degradation, lignocellulosic enzyme production and biological efficiency in Pleurotus eryngii was unclear. In this report, Pleurotus eryngii was cultivated in substrates including ramie stalks, kenaf stalks, cottonseed hulls and bulrush stalks. The results showed that ramie stalks and kenaf stalks were found to best suitable to cultivate Pleurotus eryngii with the biological efficiency achieved at 55% and 57%, respectively. In order to establish correlations between different substrates and lignocellulosic enzymes expression, the extracellular proteins from four substrates were profiled with high throughput TMT-based quantitative proteomic approach. 241 non-redundant proteins were identified and 74 high confidence lignocellulosic enzymes were quantified. Most of the cellulases, hemicellulases and lignin depolymerization enzymes were highly up-regulated when ramie stalks and kenaf stalks were used as carbon sources. The enzyme activities results suggested cellulases, hemicellulases and lignin depolymerization enzymes were significantly induced by ramie stalks and kenaf stalks. The lignocelluloses degradation, most of the lignocellulosic enzymes expressions and activities of Pleurotus eryngii had positive correlation with the biological efficiency, which depend on the nature of lignocellulosic substrates. In addition, the lignocellulosic enzymes expression profiles during Pleurotus eryngii growth in different substrates were obtained. The present study suggested that most of the lignocellulosic enzymes expressions and activities can be used as tools for selecting better performing substrates for commercial mushroom cultivation. © 2016 The Author(s) Published by S. Karger AG, Basel.

A DNA enzyme with N-glycosylase activity

NASA Technical Reports Server (NTRS)

Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

2000-01-01

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

Effects of non-starch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley.

PubMed

Li, Wei-Fen; Feng, Jie; Xu, Zi-Rong; Yang, Cai-Mei

2004-03-15

To investigate effects of non-starch polysaccharides(NSP) enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. Sixty crossbred piglets averaging 13.5 kg were randomly assigned to two treatment groups with three replications (pens) based on sex and mass. Each group was fed on the diet based on barley with or without added NSP enzymes (0.15%) for a 40-d period. At the end of the experiment the pigs were weighed. Three piglets of each group were chosen and slaughtered. Pancreas, digesta from the distal end of the duodenum and jejunal mucosa were collected for determination. Activities of the digestive enzymes trypsin, chymotrypsin, amylase and lipase were determined in the small intestinal sections as well as in homogenates of pancreatic tissue. Maltase, sucrase, lactase and gamma-glutamyl transpeptidase (gamma-GT) activities were analyzed in jejunal mucosa. Supplementation with NSP enzymes improved growth performance of piglets. It showed that NSP enzymes had no effect on digestive enzyme activities in pancreas, but decreased the activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents by 57.56%, 76.08%, 69.03% and 40.22%(P<0.05) compared with control, and increased gamma-GT activities in jejunal mucosa by 118.75%(P<0.05). Supplementation with NSP enzymes in barley based diets could improve piglets' growth performance, decrease activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents and increase gamma-GT activities in jejunal mucosa.

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. Copyright © 2011 Elsevier GmbH. All rights reserved.

Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.

PubMed

Simon, Gabriel M; Cravatt, Benjamin F

2010-04-09

Genome sequencing projects have uncovered thousands of uncharacterized enzymes in eukaryotic and prokaryotic organisms. Deciphering the physiological functions of enzymes requires tools to profile and perturb their activities in native biological systems. Activity-based protein profiling has emerged as a powerful chemoproteomic strategy to achieve these objectives through the use of chemical probes that target large swaths of enzymes that share active-site features. Here, we review activity-based protein profiling and its implementation to annotate the enzymatic proteome, with particular attention given to probes that target serine hydrolases, a diverse superfamily of enzymes replete with many uncharacterized members.

Differences between male and female rhesus monkey erythrocyte acetylcholinesterase and plasma cholinesterase activity before and after exposure to sarin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woodard, C.L.; Calamaio, C.A.; Kaminskis, A.

The female rhesus monkey has a menstrual cycle like the human. Additionally, several differences in enzyme levels between males and females and in the female during the menstrual cycle are present. Therefore we quantitated plasma cholinesterase (ChE/BuChE) and erythrocyte (RBC) acetylcholinesterase (AChE) activity before and after exposure to sarin (GB)(1 5 ug/kg, iv; a 0.75 LD50), in male and female rhesus (Macaca mulatta) monkeys. Twenty-eight-day preexposure baseline plasma ChE and RBC AChE values for six male and six female rhesus monkeys were compared for intra-animal, within sex and between sex differences. After these baseline values were obtained, the organophosphorus (OP)more » compound/Isopropyl methylphosphono-fluoridate (GB) was administered to atropinized monkeys to determine if there was a significant in vivo difference between the sexes in their response to this intoxication in regard to the rate of BuChE /AChE inhibition, pyridine-2-aldoxime methyl chloride (2-PAM) reactivation of the phosphonylated BuChE and the rate of aging of the phosphonylated:BuChE/AChE. In the pre-exposure portion of the protocol; the intra-animal and intra-group BuChE/AChE variations were found to be minimal; but there were significant differences between the male and female monkeys in both plasma BuChE and RBC AChE levels; although probably clinically insignificant in respect to an OP intoxication. No significant cyclic fluctuations were seen during the 28-day study in either sex.« less

Micropollutant degradation via extracted native enzymes from activated sludge.

PubMed

Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

2016-05-15

A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

Development of Activity-based Cost Functions for Cellulase, Invertase, and Other Enzymes

NASA Astrophysics Data System (ADS)

Stowers, Chris C.; Ferguson, Elizabeth M.; Tanner, Robert D.

As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

[125I]Iodo-ASEM, a specific in vivo radioligand for α7-nAChR

PubMed Central

Gao, Yongjun; Mease, Ronnie C.; Olson, Thao T.; Kellar, Kenneth J.; Dannals, Robert F.; Pomper, Martin G.; Horti, Andrew G.

2014-01-01

[125I]Iodo-ASEM, a new radioligand with high affinity and selectivity for α7-nAChRs (Ki = 0.5 nM; α7/α4β2 = 3,414), has been synthesized in radiochemical yield of 33 ± 6% from the corresponding di-butyltriazene derivative and at high specific radioactivity (1,600 Ci/mmol; 59.2 MBq/μmol). [125I]Iodo-ASEM readily entered the brains of normal CD-1 mice and specifically and selectively labeled cerebral α7-nAChRs. [125I]iodo-ASEM is a new useful tool for studying α7-nAChR. PMID:25687449

Glutathione Peroxidase Enzyme Activity in Aging

PubMed Central

Espinoza, Sara E.; Guo, Hongfei; Fedarko, Neal; DeZern, Amy; Fried, Linda P.; Xue, Qian-Li; Leng, Sean; Beamer, Brock; Walston, Jeremy D.

2010-01-01

Background It is hypothesized that free radical damage contributes to aging. Age-related decline in activity of the antioxidant enzyme glutathione peroxidase (GPx) may contribute to increased free radicals. We hypothesized that GPx activity decreases with age in a population of older women with disability. Methods Whole blood GPx activity was measured in baseline stored samples from participants in the Women's Health and Aging Study I, a cohort of disabled community-dwelling older women. Linear regression was used to determine cross-sectional associations between GPx activity and age, adjusting for hemoglobin, coronary disease, diabetes, selenium, and body mass index. Results Six hundred one participants had complete demographic, disease, and laboratory information. An inverse association was observed between GPx and age (regression coefficient = −2.9, p < .001), indicating that for each 1-year increase in age, GPx activity decreased by 2.9 μmol/min/L. This finding remained significant after adjustment for hemoglobin, coronary disease, diabetes, and selenium, but not after adjustment for body mass index and weight loss. Conclusion This is the first study to examine the association between age and GPx activity in an older adult cohort with disability and chronic disease. These findings suggest that, after age 65, GPx activity declines with age in older women with disability. This decline does not appear to be related to diseases that have been previously reported to alter GPx activity. Longitudinal examination of GPx activity and other antioxidant enzymes in diverse populations of older adults will provide additional insight into age- and disease-related changes in these systems. PMID:18511755

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

PubMed Central

Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

Structure—activity relationships for insecticidal carbamates*

PubMed Central

Metcalf, Robert L.

1971-01-01

Carbamate insecticides are biologically active because of their structural complementarity to the active site of acetylcholinesterase (AChE) and their consequent action as substrates with very low turnover numbers. Carbamates behave as synthetic neurohormones that produce their toxic action by interrupting the normal action of AChE so that acetylcholine accumulates at synaptic junctions. The necessary properties for a suitable insecticidal carbamate are lipid solubility, suitable structural complementarity to AChE, and sufficient stability to multifunction-oxidase detoxification. The relationships between the structure and the activity of a large number of synthetic carbamates are analysed in detail, with particular attention to the second of these properties. PMID:5315358

The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies.

PubMed

Muldoon, P P; Jackson, K J; Perez, E; Harenza, J L; Molas, S; Rais, B; Anwar, H; Zaveri, N T; Maldonado, R; Maskos, U; McIntosh, J M; Dierssen, M; Miles, M F; Chen, X; De Biasi, M; Damaj, M I

2014-08-01

Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. © 2014 The British Pharmacological Society.