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.

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.

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

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

PubMed Central

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

2013-01-01

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

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.

High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts.

PubMed

de Jong, Camiel F; Derks, Rico J E; Bruyneel, Ben; Niessen, Wilfried; Irth, Hubertus

2006-04-21

The present paper describes a High-performance liquid chromatography-mass spectrometry (LC-MS) methodology for the screening of acetylcholinesterase (AChE) inhibitors in natural extracts. AChE activity of sample components is monitored by a post-column biochemical assay that is based on the separate, sequential mixing of AChE and acetylcholine, respectively, with the HPLC eluate. AChE inhibitors are detected by measuring a decrease of product formation using electrospray MS. Ammonium bicarbonate was used as buffer in order to achieve optimum compatibility between biochemical assay and MS detection conditions. The assay is robust and stable for over 13 h and compares favourably with other AChE assays in terms of stability and sensitivity. IC(50) values of 9-aminoacridine, galanthamine, gallamine, (-)-huperzine A and thioflavin T were determined to be 0.12, 0.38, 6.4, 0.46 and 3.2 microM, respectively. The assay was used to effectively identify an AChE inhibitor present in a crude extract of Narcissus c.v. "Bridal Crown".

[Interest of the cholinesterase assay during organophosphate poisonings].

PubMed

Jalady, A-M; Dorandeu, F

2013-12-01

Cholinesterases are the main targets of organophosphorus compounds. The two enzymes present in the blood (butyrylcholinesterase, BChE; acetylcholinesterase, AChE) are biomarkers of their systemic toxicity. Activity of the plasma BChE is very often determined as it allows a rapid diagnostic of poisoning and is a marker of the persistence of the toxicant in the blood. The activity of the red blood cell AChE gives a better picture of the synaptic inhibition in the nervous system but the assay is less commonly available in routine laboratories. Better biomarker of the exposure, it allows a diagnosis of the severity of the poisoning and helps to assess the efficacy of oxime therapy. Besides the practical aspects of blood collection and sample processing, and the interpretation of the assays, this review stresses the complementarity of both enzyme assays and recalls their crucial interest for the confirmation of poisoning with an organophosphorus in a situation of war or terrorist attack and for the monitoring of occupational exposures. Copyright © 2013. Published by Elsevier SAS.

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.

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

PubMed

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

2014-09-01

Effects of glyphosate based herbicide, Excel Mera 71 at a dose of 17.20mg/l on enzyme activities of acetylcholinesterase (AChE), lipid peroxidation (LPO), catalase (CAT), glutathione-S-transferase (GST) and protein content were measured in different tissues of two Indian air-breathing teleosts, Anabas testudineus (Bloch) and Heteropneustes fossilis (Bloch) during an exposure period of 30 days under laboratory condition. AChE activity was significantly increased in all the investigated tissues of both fish species and maximum elevation was observed in brain of H. fossilis, while spinal cord of A. testudineus showed minimum increment. Fishes showed significant increase LPO levels in all the tissues; highest was observed in gill of A. testudineus but lowest LPO level was observed in muscle of H. fossilis. CAT was also enhanced in both the fishes, while GST activity in liver diminished substantially and minimum was observed in liver of A. testudineus. Total protein content showed decreased value in all the tissues, maximum reduction was observed in liver and minimum in brain of A. testudineus and H. fossilis respectively. The results indicated that Excel Mera 71 caused serious alterations in the enzyme activities resulting into severe deterioration of fish health; so, AChE, LPO, CAT and GST can be used as suitable indicators of herbicidal toxicity. Copyright © 2014 Elsevier Inc. All rights reserved.

Tertiary amine derivatives of chlorochalcone as acetylcholinesterase (AChE) and buthylcholinesterase (BuChE) inhibitors: the influence of chlorine, alkyl amine side chain and α,β-unsaturated ketone group.

PubMed

Gao, Xiao-Hui; Zhou, Chao; Liu, Hao-Ran; Liu, Lin-Bo; Tang, Jing-Jing; Xia, Xin-Hua

2017-12-01

A new series of tertiary amine derivatives of chlorochalcone (4a∼4l) were designed, synthesized and evaluated for the effect on acetylcholinesterase (AChE) and buthylcholinesterase (BuChE). The results indicated that all compounds revealed moderate or potent inhibitory activity against AChE, and some possessed high selectivity for AChE over BuChE. The structure-activity investigation showed that the substituted position of chlorine significantly influenced the activity and selectivity. The alteration of tertiary amine group also leads to obvious change in bioactivity. Among them, IC 50 of compound 4l against AChE was 0.17 ± 0.06 µmol/L, and the selectivity was 667.2 fold for AChE over BuChE. Molecular docking and enzyme kinetic study on compound 4l suggested that it simultaneously binds to the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. Further study showed that the pyrazoline derivatives synthesized from chlorochalcones had weaker activity and lower selectivity in inhibiting AChE compared to that of chlorochalcone derivatives.

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

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.

Novel potent pyridoxine-based inhibitors of AChE and BChE, structural analogs of pyridostigmine, with improved in vivo safety profile.

PubMed

Strelnik, Alexey D; Petukhov, Alexey S; Zueva, Irina V; Zobov, Vladimir V; Petrov, Konstantin A; Nikolsky, Evgeny E; Balakin, Konstantin V; Bachurin, Sergey O; Shtyrlin, Yurii G

2016-08-15

We report a novel class of carbamate-type ChE inhibitors, structural analogs of pyridostigmine. A small library of congeneric pyridoxine-based compounds was designed, synthesized and evaluated for AChE and BChE enzymes inhibition in vitro. The most active compounds have potent enzyme inhibiting activity with IC50 values in the range of 0.46-2.1μM (for AChE) and 0.59-8.1μM (for BChE), with moderate selectivity for AChE comparable with that of pyridostigmine and neostigmine. Acute toxicity studies using mice models demonstrated excellent safety profile of the obtained compounds with LD50 in the range of 22-326mg/kg, while pyridostigmine and neostigmine are much more toxic (LD50 3.3 and 0.51mg/kg, respectively). The obtained results pave the way to design of novel potent and safe cholinesterase inhibitors for symptomatic treatment of neuromuscular disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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.

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.

Polyacrylic acid-coated cerium oxide nanoparticles: An oxidase mimic applied for colorimetric assay to organophosphorus pesticides.

PubMed

Zhang, Shi-Xiang; Xue, Shi-Fan; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

2016-11-15

It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs. Copyright © 2016 Elsevier B.V. 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.

Unbinding of fluorinated oxime drug from the AChE gorge in polarizable water: a well-tempered metadynamics study.

PubMed

Pathak, Arup Kumar; Bandyopadhyay, Tusar

2017-02-15

Despite the fact that fluorination makes a drug more lipophilic, the molecular level understanding of protein-fluorinated drug interactions is very poor. Due to their enhanced ability to penetrate the blood brain barrier, they are suitable for reactivation of organophosphorus inactivated acetylcholinesterase (AChE) in the central nervous system. We systematically studied the unbinding of fluorinated obidoxime (FOBI) and non-fluorinated obidoxime (OBI) from the active site gorge of the serine hydrolase AChE in mean field polarizable water by employing all atom molecular dynamics simulations. It is observed that the unbinding process is strongly influenced by cation-π, hydrogen bond (HB) and water bridge interactions. The FOBI drug interacts more strongly with the protein residues than OBI and this is also verified from quantum mechanical calculations. Distinct unbinding pathways for FOBI and OBI are observed as evident from the 1D and 2D potential of mean force of the unbinding profiles. The present study suggests that the FOBI drug is held more firmly in the gorge of AChE in comparison to OBI and may lead to higher reactivation efficiency of the inactivated enzyme.

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.

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

A new continuous fluorometric assay for acetylcholinesterase activity and inhibitor screening with emissive core-shell silica particles containing tetraphenylethylene fluorophore.

PubMed

Shen, Xiang; Liang, Fuxin; Zhang, Guanxin; Zhang, Deqing

2012-05-07

Emissive core-shell silica particles with tetraphenylethylene moieties were prepared and characterized. Fluorescence quenching was observed for the silica particles upon addition of compound 2 (Dabcyl-ACh). This was attributed to the electrostatic interaction between the silica particles and 2 and the resulting photoinduced energy transfer between them. After incubation with AChE, the fluorescence intensity started to increase. The fluorescence enhancement became more significant when the concentration of AChE was higher. The reaction kinetic parameters for AChE were successfully estimated with the silica particles and 2. These results reveal that the ensemble of the silica particles and 2 can be utilized for AChE assay. Moreover, the fluorescence spectra of the ensemble of the silica particles and 2 containing AChE were also measured after further addition of either neostigmine or tacrine which are typical inhibitors of AChE. The results manifest that the ensemble of the emissive silica particles and 2 is also useful for screening the inhibitors of AChE.

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.

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

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

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

Specific radioisotopic assay for cholinesterase

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

Talbot, B.G.; Anderson, D.R.; Harris, L.W.

1992-02-01

The authors have developed a radiometric method (1) for measuring cholinesterase (ChE) activity that uses 0.4 N perchloric acid (PCA), instead of p-dioxane (method 2), to denature the ChE and stop hydrolysis of acetyl-1-{sup 14}C-choline (ACh). The unreacted ACh is removed by IRP-69 cationic exchange resin suspended in water, instead of dioxane. The supernatant containing the hydrolysis product, {sup 14}C-acetic acid, is mixed with nonhazardous scintillation cocktail and counted. The incubation mixture of 1 is similar to 2 and contains 0.1 ml of buffer-salt solution 0.1 ml of guinea pig whole blood (WB)-water suspension and 0.1 ml of 3 mMmore » ACh solution. Procedures 1 and 2 were compared to a titragraphic assay for ChE activity; specific activity values of WB (mmol ACh hydrolyzed/ml/hr) were found to be 137.6, 72.4 and 135.0, respectively. When {sup 14}C-acetic acid was processed through procedures 1 and 2, significantly less {sup 14}C was found in the supernatant from 2, whereas all of the expected {sup 14}C was found in the supernatant from 1, suggesting that IRP-69 resin in dioxane will remove significant amounts of {sup 14}C-acetic acid.« less

Understanding the conformational flexibility and electrostatic properties of curcumin in the active site of rhAChE via molecular docking, molecular dynamics, and charge density analysis.

PubMed

Saravanan, Kandasamy; Kalaiarasi, Chinnasamy; Kumaradhas, Poomani

2017-12-01

Acetylcholinesterase (AChE) is an important enzyme responsible for Alzheimer's disease, as per report, keto-enol form of curcumin inhibits this enzyme. The present study aims to understand the binding mechanism of keto-enol curcumin with the recombinant human Acetylcholinesterase (rhAChE) from its conformational flexibility, intermolecular interactions, charge density distribution, and the electrostatic properties at the active site of rhAChE. To accomplish this, a molecular docking analysis of curcumin with the rhAChE was performed, which gives the structure and conformation of curcumin in the active site of rhAChE. Further, the charge density distribution and the electrostatic properties of curcumin molecule (lifted from the active site of rhAChE) were determined from the high level density functional theory (DFT) calculations coupled with the charge density analysis. On the other hand, the curcumin molecule was optimized (gas phase) using DFT method and further, the structure and charge density analysis were also carried out. On comparing the conformation, charge density distribution and the electrostatic potential of the active site form of curcumin with the corresponding gas phase form reveals that the above said properties are significantly altered when curcumin is present in the active site of rhAChE. The conformational stability and the interaction of curcumin in the active site are also studied using molecular dynamics simulation, which shows a large variation in the conformational geometry of curcumin as well as the intermolecular interactions.

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.

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.

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

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.

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

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

Dual enzyme activities assay by quantitative electrospray ionization quadrupole-time-of-flight mass spectrometry.

PubMed

Cai, Tingting; Zhang, Li; Wang, Haoyang; Zhang, Jing; Wang, Rong; Zhang, Yurong; Guo, Yinlong

2012-01-01

A practical and rapid method based on electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-ToF MS) was developed for detecting activities of both acetylcholinesterase IAChEI and glutathione S-transferase (GST). The simultaneous study of these two enzyme activities is significant for studying human bio-functions, especially for those who take in toxic compounds and have a risk of disease. Here, the enzyme activities were represented by the conversion of enzymatic substrates and determined by quantitatively analyzing enzymatic substrates. Different internal standards were used to quantify each enzymatic substrate and the good linearity of calibration curves demonstrated the feasibility of the internal standards. The Michaelis-Menten constants (Km) of both GST and AChE were measured by this method and were consistent with values previously reported. Furthermore, we applied this approach to detect GST and AChE activities of whole bloods from four deceased and healthy people. The variation in enzyme activity was in accord with information from gas chromatography mass spectrometry [GC/MS). The screening of AChE and GST provided reliable results and strong forensic evidence. This method offers an alternative choice for detecting enzyme activities and is anticipated to have wide applications in pharmaceutical research and prevention in toxic compounds.

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.

HIGHLY SENSITIVE ASSAY FOR ANTICHOLINESTERASE COMPOUNDS USING 96 WELL PLATE FORMAT

EPA Science Inventory

The rapid and sensitive detection of organophosphate insecticides using a 96 well plate format is reported. Several features of this assay make it attractive for development as a laboratory-based or field screening assay. Acetylcholinesterase (AChE) was stabilized in a gelati...

A smartphone-readable barcode assay for the detection and quantitation of pesticide residues.

PubMed

Guo, Juan; Wong, Jessica X H; Cui, Caie; Li, Xiaochun; Yu, Hua-Zhong

2015-08-21

In this paper, we present a smartphone-readable barcode assay for the qualitative detection of methyl parathion residues, a toxic organophosphorus pesticide that is popularly used in agriculture worldwide. The detection principle is based on the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AchE) by methyl parathion; AchE catalytically hydrolyzes acetylthiocholine iodine to thiocholine that in turn dissociates dithiobis-nitrobenzoate to produce a yellow product (deprotonated thio-nitrobenzoate). The yellow intensity of the product was confirmed to be inversely dependent on the concentration of the pesticide. We have designed a barcode-formatted assay chip by using a PDMS (polydimethylsiloxane) channel plate (as the reaction reservoir), situated under a printed partial barcode, to complete the whole barcode such that it can be directly read by a barcode scanning app installed on a smartphone. The app is able to qualitatively present the result of the pesticide test; the absence or a low concentration of methyl parathion results in the barcode reading as "-", identifying the test as negative for pesticides. Upon obtaining a positive result (the app reads a "+" character), the captured image can be further analyzed to quantitate the methyl parathion concentration in the sample. Besides the portability and simplicity, this mobile-app based colorimetric barcode assay compares favorably with the standard spectrophotometric method.

Bioprofiling of Salvia miltiorrhiza via planar chromatography linked to (bio)assays, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

PubMed

Azadniya, Ebrahim; Morlock, Gertrud E

2018-01-19

An affordable bioanalytical workflow supports the collection of data on active ingredients, required for the understanding of health-related food, superfood and traditional medicines. Targeted effect-directed responses of single compounds in a complex sample highlight this powerful bioanalytical hyphenation of planar chromatography with (bio)assays. Among many reports about biological properties of Salvia miltiorrhiza Bunge root (Danshen) and their analytical methods, the highly efficient direct bioautography (DB) workflow has not been considered so far. There was just one TLC-acetylcholinesterase (AChE) method with a poor zone resolution apart from our two HPTLC-DB studies, however, all methods were focused on the nonpolar extracts of Danshen (tanshinones) only. The current study on HPTLC-UV/Vis/FLD-(bio)assay-HRMS, followed by streamlined scale-up to preparative layer chromatography (PLC)- 1 H-NMR, aimed at an even more streamlined, yet comprehensive bioanalytical workflow. It comprised effect-directed screening of both, its polar (containing phenolics) and nonpolar extracts (containing tanshinones) on the same HPTLC plate, the biochemical and biological profiling with four different (bio)assays and elucidation of structures of known and unidentified active compounds. The five AChE inhibitors, salvianolic acid B (SAB), lithiospermic acid (LSA) and rosmarinic acid (RA) as well as cryptotanshinone (CT) and 15,16-dihydrotanshinone I (DHTI) were confirmed, but also unidentified inhibitors were observed. In the polar extracts, SAB, LSA and RA exhibited free radical scavenging properties in the 2,2-diphenyl-1-picrylhydrazyl assay. CT, DHTI and some unidentified nonpolar compounds were found active against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri (LOD 12 ng/band for CT, and 5 ng/band for DHTI). For the first time, the most multipotent unidentified active compound zone in the B. subtilis, A. fischeri and AChE fingerprints of the nonpolar

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).

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

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.

Menthol Enhances Nicotine Reward-Related Behavior by Potentiating Nicotine-Induced Changes in nAChR Function, nAChR Upregulation, and DA Neuron Excitability.

PubMed

Henderson, Brandon J; Wall, Teagan R; Henley, Beverley M; Kim, Charlene H; McKinney, Sheri; Lester, Henry A

2017-11-01

Understanding why the quit rate among smokers of menthol cigarettes is lower than non-menthol smokers requires identifying the neurons that are altered by nicotine, menthol, and acetylcholine. Dopaminergic (DA) neurons in the ventral tegmental area (VTA) mediate the positive reinforcing effects of nicotine. Using mouse models, we show that menthol enhances nicotine-induced changes in nicotinic acetylcholine receptors (nAChRs) expressed on midbrain DA neurons. Menthol plus nicotine upregulates nAChR number and function on midbrain DA neurons more than nicotine alone. Menthol also enhances nicotine-induced changes in DA neuron excitability. In a conditioned place preference (CPP) assay, we observed that menthol plus nicotine produces greater reward-related behavior than nicotine alone. Our results connect changes in midbrain DA neurons to menthol-induced enhancements of nicotine reward-related behavior and may help explain how smokers of menthol cigarettes exhibit reduced cessation rates.

Menthol Enhances Nicotine Reward-Related Behavior by Potentiating Nicotine-Induced Changes in nAChR Function, nAChR Upregulation, and DA Neuron Excitability

PubMed Central

Henderson, Brandon J; Wall, Teagan R; Henley, Beverley M; Kim, Charlene H; McKinney, Sheri; Lester, Henry A

2017-01-01

Understanding why the quit rate among smokers of menthol cigarettes is lower than non-menthol smokers requires identifying the neurons that are altered by nicotine, menthol, and acetylcholine. Dopaminergic (DA) neurons in the ventral tegmental area (VTA) mediate the positive reinforcing effects of nicotine. Using mouse models, we show that menthol enhances nicotine-induced changes in nicotinic acetylcholine receptors (nAChRs) expressed on midbrain DA neurons. Menthol plus nicotine upregulates nAChR number and function on midbrain DA neurons more than nicotine alone. Menthol also enhances nicotine-induced changes in DA neuron excitability. In a conditioned place preference (CPP) assay, we observed that menthol plus nicotine produces greater reward-related behavior than nicotine alone. Our results connect changes in midbrain DA neurons to menthol-induced enhancements of nicotine reward-related behavior and may help explain how smokers of menthol cigarettes exhibit reduced cessation rates. PMID:28401925

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.

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.

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

and/or targets. We have carried out some theoretical-experimental studies to illustrate the practical use of 3D MI-DRAGON. First, we have reported the prediction and pharmacological assay of 22 different rasagiline derivatives with possible AChE inhibitory activity. In this work, we have reviewed different computational studies on Drug- Protein models. First, we have reviewed 10 studies on DP computational models. Next, we have reviewed 2D QSAR, 3D QSAR, CoMFA, CoMSIA and Docking with different compounds to find Drug-Protein QSAR models. Last, we have developped a 3D multi-target QSAR (3D mt-QSAR) models for the prediction of the activity of new compounds against different targets or the discovery of new targets.

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.

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

USDA-ARS?s Scientific Manuscript database

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

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.

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

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.

Geissoschizine methyl ether N-oxide, a new alkaloid with antiacetylcholinesterase activity from Uncaria rhynchophylla.

PubMed

Jiang, Wei-Wei; Su, Jia; Wu, Xing-De; He, Juan; Peng, Li-Yan; Cheng, Xiao; Zhao, Qin-Shi

2015-01-01

Geissoschizine methyl ether N-oxide, a new oxindole alkaloid, along with 14 known alkaloids, was isolated from the aerial part of Uncaria rhynchophylla. Their structures were identified by comprehensive spectral methods, including 2D NMR experiments, and confirmed by comparing with the literature data. In vitro acetylcholinesterase (AChE) inhibitory activity assay showed that the new compound exhibited anti-AChE activity with IC₅₀ value of 23.4 μM.

Screening for acetylcholinesterase inhibition and antioxidant activity of selected plants from Croatia.

PubMed

Jukic, Mila; Burcul, Franko; Carev, Ivana; Politeo, Olivera; Milos, Mladen

2012-01-01

The methanol, ethyl acetate and chloroform extracts of selected Croatian plants were tested for their acetylcholinesterase (AChE) inhibition and antioxidant activity. Assessment of AChE inhibition was carried out using microplate reader at 1 mg mL⁻¹. Antioxidant capacities were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test and ferric reducing/antioxidant power assay (FRAP). Total phenol content (TPC) of extracts were determined using Folin-Ciocalteu colorimetric method. Out of 48 extracts, only methanolic extract of the Salix alba L. cortex exerted modest activity towards AChE, reaching 50.80% inhibition at concentration of 1 mg mL⁻¹. All the other samples tested had activity below 20%. The same extract performed the best antioxidative activity using DPPH and FRAP method, too. In essence, among all extracts used in the screening, methanolic extracts showed the best antioxidative activity as well as highest TPC.

Insights into cholinesterase inhibitory and antioxidant activities of five Juniperus species.

PubMed

Orhan, Nilufer; Orhan, Ilkay Erdogan; Ergun, Fatma

2011-09-01

In vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory and antioxidant activities of the aqueous and ethanol extracts of the leaves, ripe fruits, and unripe fruits of Juniperus communis ssp. nana, Juniperus oxycedrus ssp. oxycedrus, Juniperus sabina, Juniperus foetidissima, and Juniperus excelsa were investigated in the present study. Cholinesterase inhibition of the extracts was screened using ELISA microplate reader. Antioxidant activity of the extracts was tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging, ferrous ion-chelating, and ferric-reducing antioxidant power (FRAP) assays. Total phenol and flavonoid contents of the extracts were determined spectrophotometrically. The extracts had low or no inhibition towards AChE, whereas the leaf aqueous extract of J. foetidissima showed the highest BChE inhibition (93.94 ± 0.01%). The leaf extracts usually exerted higher antioxidant activity. We herein describe the first study on anticholinesterase and antioxidant activity by the methods of ferrous ion-chelating, superoxide radical scavenging, and ferric-reducing antioxidant power (FRAP) assays of the mentioned Juniperus species. Copyright © 2011 Elsevier Ltd. All rights reserved.

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.

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.

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

Compounds from the aerial parts of Piper bavinum and their anti-cholinesterase activity.

PubMed

Dung, Hoang Viet; Cuong, To Dao; Chinh, Nguyen Minh; Quyen, Do; Kim, Jeong Ah; Byeon, Jeong Su; Woo, Mi Hee; Choi, Jae Sui; Min, Byung Sun

2015-01-01

A new alkenylphenol, bavinol A (1), together with six known compounds (2-7) were isolated from the aerial parts of Piper bavinum (Piperaceae). The chemical structures of these compounds were determined by spectroscopic analyses including 2D NMR spectroscopy. The anti-Alzheimer effects of compounds 1-7 were evaluated from acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity assays. Bavinol A (1), ampelopsin (3), and violanthin (4) exhibited AChE inhibitory activities with IC50 values of 29.80, 59.47 and 79.80 μM. Compound 1 also showed the most potent BChE inhibitory activity with an IC50 value of 19.25 μM.

Study of acetylcholinesterase activity and apoptosis in SH-SY5Y cells and mice exposed to ethanol.

PubMed

Sun, Wenjun; Chen, Liangjing; Zheng, Wei; Wei, Xiaoan; Wu, Wenqi; Duysen, Ellen G; Jiang, Wei

2017-06-01

Ethanol is one of the most commonly abused psychotropic substances with deleterious effects on the central nervous system. Ethanol exposure during development results in the loss of neurons in brain regions and when exposed to ethanol cultured cells undergo apoptosis. To date no information is available on whether abnormally high AChE activity is characteristic of apoptosis in animals exposed to ethanol. The aims of the present study were to determine whether induction of AChE activity is associated with ethanol-induced apoptosis and to explore the mechanism of enhanced AChE activity induced by ethanol. For this purpose, in vitro and in vivo experiments were performed. AChE activity was quantified by spectrophotometry and apoptosis by flow cytometer in SH-SY5Y cells exposed to ethanol. The results showed that cells treated with 500mM ethanol for 24h had a 9-fold increase in apoptotic cells and a 6-fold increase in AChE activity compared with controls. Mice exposed acutely to 200μl of 20% ethanol daily on days 1-4 had elevated AChE activity in plasma on days 3-7. On day 4, plasma AChE activity was 2.4-fold higher than pretreatment activity. More apoptotic cells were found in the brains of treated mice compared to controls. Cells in brain sections that were positive in the TUNEL assay stained for AChE activity. In conclusion, AChE activity and apoptosis were induced in SH-SY5Y cells and mice treated with ethanol, which may indicate that increased AChE may related to apoptosis induced by ethanol. Unusually high AChE activity may be an effect marker of exposure to ethanol. The relationship between AChE and apoptosis might represent a novel mechanism of ethanol-associated neuronal injury. Copyright © 2017 Elsevier B.V. All rights reserved.

Anticholinesterase inhibitory activity of quaternary alkaloids from Tinospora crispa.

PubMed

Yusoff, Mashitah; Hamid, Hazrulrizawati; Houghton, Peter

2014-01-20

Quaternary alkaloids are the major alkaloids isolated from Tinospora species. A previous study pointed to the necessary presence of quaternary nitrogens for strong acetylcholinesterase (AChE) inhibitory activity in such alkaloids. Repeated column chromatography of the vine of Tinospora crispa extract led to the isolation of one new protoberberine alkaloid, 4,13-dihydroxy-2,8,9-trimethoxydibenzo[a,g]quinolizinium (1), along with six known alkaloids-dihydrodiscretamine (2), columbamine (3), magnoflorine (4), N-formylannonaine (5), N-formylnornuciferine (6), and N-trans-feruloyltyramine (7). The seven compounds were isolated and structurally elucidated by spectroscopic analysis. Two known alkaloids, namely, dihydrodiscretamine and columbamine are reported for the first time for this plant. The compounds were tested for AChE inhibitory activity using Ellman's method. In the AChE inhibition assay, only columbamine (3) showed strong activity with IC50 48.1 µM. The structure-activity relationships derived from these results suggest that the quaternary nitrogen in the skeleton has some effect, but that a high degree of methoxylation is more important for acetylcholinesterase inhibition.

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

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland.

PubMed

Ishikawa, Yasuko; Yuan, Zhenfang; Inoue, Noriko; Skowronski, Mariusz T; Nakae, Yoshiko; Shono, Masayuki; Cho, Gota; Yasui, Masato; Agre, Peter; Nielsen, Søren

2005-11-01

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M(3) muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M(3) mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca(2+) signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.

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

M1/M2 muscarinic receptor selectivity using potassium (K/sup +/)-stimulated release of (/sup 3/H)-dopamine (DA) and (/sup 14/C)-acetyl-choline (ACH) in striatum

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

DeHaven, D.L.; Steranka, L.R.

Raiteri et al have suggested that muscarinic receptor subtypes can be differentiated in striatal synaptosomes by the release of DA (M1) or ACh (M2). The authors attempted to replicate this finding and to characterize responses of selective and non-selective cholinergic agonists and antagonists using K+-stimulated release of transmitters from rat striatal slices. The non-selective agonists ACh, carbachol and oxotremorine stimulated release of (/sup 3/H)-DA and inhibited release of (/sup 14/C)-ACh with EC50 values of 10.6, 9.2 and 4.2 ..mu..M (DA) and 1.2, 0.77 and 0.43 ..mu..M (ACh), respectively. The M1 agonist McN-A-343-11 selectively inhibited release of DA with an EC50more » value of 4.8 ..mu..M. Pilocarpine was ineffective in this system. The M1 antagonist pirenzepine reversed the effects of 10/sup -4/ M carbachol on release with an eight-fold selectivity for release of (/sup 3/H)-DA (IC50 = 0.77 ..mu..M) vs (/sup 14/C)-ACh (IC50 = 6.3 ..mu..M). These results suggest that although this system can determine relative subtype selectivities, the results obtained in this assay do not always correlate with those obtained from phosphatidyl inositol turnover or adenylate cyclase activity.« less

An Acetylcholinesterase-Based Chronoamperometric Biosensor for Fast and Reliable Assay of Nerve Agents

PubMed Central

Pohanka, Miroslav; Adam, Vojtech; Kizek, Rene

2013-01-01

The enzyme acetylcholinesterase (AChE) is an important part of cholinergic nervous system, where it stops neurotransmission by hydrolysis of the neurotransmitter acetylcholine. It is sensitive to inhibition by organophosphate and carbamate insecticides, some Alzheimer disease drugs, secondary metabolites such as aflatoxins and nerve agents used in chemical warfare. When immobilized on a sensor (physico-chemical transducer), it can be used for assay of these inhibitors. In the experiments described herein, an AChE- based electrochemical biosensor using screen printed electrode systems was prepared. The biosensor was used for assay of nerve agents such as sarin, soman, tabun and VX. The limits of detection achieved in a measuring protocol lasting ten minutes were 7.41 × 10−12 mol/L for sarin, 6.31 × 10−12 mol/L for soman, 6.17 × 10−11 mol/L for tabun, and 2.19 × 10−11 mol/L for VX, respectively. The assay was reliable, with minor interferences caused by the organic solvents ethanol, methanol, isopropanol and acetonitrile. Isopropanol was chosen as suitable medium for processing lipophilic samples. PMID:23999806

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.

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.

Distribution of intravenously administered acetylcholinesterase inhibitor and acetylcholinesterase activity in the adrenal gland: 11C-donepezil PET study in the normal rat.

PubMed

Watabe, Tadashi; Naka, Sadahiro; Ikeda, Hayato; Horitsugi, Genki; Kanai, Yasukazu; Isohashi, Kayako; Ishibashi, Mana; Kato, Hiroki; Shimosegawa, Eku; Watabe, Hiroshi; Hatazawa, Jun

2014-01-01

Acetylcholinesterase (AChE) inhibitors have been used for patients with Alzheimer's disease. However, its pharmacokinetics in non-target organs other than the brain has not been clarified yet. The purpose of this study was to evaluate the relationship between the whole-body distribution of intravenously administered (11)C-Donepezil (DNP) and the AChE activity in the normal rat, with special focus on the adrenal glands. The distribution of (11)C-DNP was investigated by PET/CT in 6 normal male Wistar rats (8 weeks old, body weight  = 220 ± 8.9 g). A 30-min dynamic scan was started simultaneously with an intravenous bolus injection of (11)C-DNP (45.0 ± 10.7 MBq). The whole-body distribution of the (11)C-DNP PET was evaluated based on the Vt (total distribution volume) by Logan-plot analysis. A fluorometric assay was performed to quantify the AChE activity in homogenized tissue solutions of the major organs. The PET analysis using Vt showed that the adrenal glands had the 2nd highest level of (11)C-DNP in the body (following the liver) (13.33 ± 1.08 and 19.43 ± 1.29 ml/cm(3), respectively), indicating that the distribution of (11)C-DNP was the highest in the adrenal glands, except for that in the excretory organs. The AChE activity was the third highest in the adrenal glands (following the small intestine and the stomach) (24.9 ± 1.6, 83.1 ± 3.0, and 38.5 ± 8.1 mU/mg, respectively), indicating high activity of AChE in the adrenal glands. We demonstrated the whole-body distribution of (11)C-DNP by PET and the AChE activity in the major organs by fluorometric assay in the normal rat. High accumulation of (11)C-DNP was observed in the adrenal glands, which suggested the risk of enhanced cholinergic synaptic transmission by the use of AChE inhibitors.

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

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.

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.

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

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.

Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures.

PubMed

Di Liberto, V; Borroto-Escuela, D O; Frinchi, M; Verdi, V; Fuxe, K; Belluardo, N; Mudò, G

2017-02-01

Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons. Western blotting and in situ proximity ligation assay (PLA) were used to detect FGFR phosphorylation (pFGFR) levels and M 1 R-FGFR1 heteroreceptor complexes, respectively. Oxotremorine-M, a non-selective mAChRs agonist, was able to transactivate FGFR and this transactivation was blocked by Src inhibitors. Oxotremorine-M treatment produced a significant increase in the primary neurite outgrowth that was blocked by pre-treatment with the pFGFR inhibitor SU5402 and Src inhibitors. This trophic effect was almost similar to that induced by fibroblast growth factor-2 (FGF-2). By using atropine as nonselective mAChRs or pirenzepine as selective antagonist for M 1 receptor (M 1 R) we could show that mAChRs are involved in modulating the pFGFRs. Using PLA, M 1 R-FGFR1 heteroreceptor complexes were identified in the hippocampus and cerebral cortex. The current findings, by showing functional mAChR-FGFR interactions, will contribute to advance the understanding of the mechanisms involved in the actions of cholinergic drugs on neuronal plasticity. Data may help to develop novel therapeutic strategies not only for neurodegenerative diseases but also for depression-induced atrophy of hippocampal neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

New derivatives of 3,4-dihydroisoquinoline-3-carboxylic acid with free-radical scavenging, D-amino acid oxidase, acetylcholinesterase and butyrylcholinesterase inhibitory activity.

PubMed

Solecka, Jolanta; Guśpiel, Adam; Postek, Magdalena; Ziemska, Joanna; Kawęcki, Robert; Lęczycka, Katarzyna; Osior, Agnieszka; Pietrzak, Bartłomiej; Pypowski, Krzysztof; Wyrzykowska, Agata

2014-09-30

A series of 3,4-dihydroisoquinoline-3-carboxylic acid derivatives were synthesised and tested for their free-radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS·+), superoxide anion radical (O2·-) and nitric oxide radical (·NO) assays. We also studied d-amino acid oxidase (DAAO), acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity. Almost each of newly synthesised compounds exhibited radical scavenging capabilities. Moreover, several compounds showed moderate inhibitory activities against DAAO, AChE and BuChE. Compounds with significant free-radical scavenging activity may be potential candidates for therapeutics used in oxidative-stress-related diseases.

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.

Phytochemical compositions of extract from peel of hawthorn fruit, and its antioxidant capacity, cell growth inhibition, and acetylcholinesterase inhibitory activity.

PubMed

Wu, Panpan; Li, Fajie; Zhang, Jianyong; Yang, Bin; Ji, Zhaojie; Chen, Weidong

2017-03-11

Hawthorn fruit (HF) is a well-known traditional medicine in China with the effects of improving digestion and regulating qi-flowing for removing blood stasis. Modern pharmacological experiments showed that HF extract has various pharmaceutical properties and flavonoids are considered as the main bioactive compounds. In this paper, Diaion HP-20 adsorption chromatography was used to enrich flavonoids in PHF, and the phytochemical composition of EPHF was analyzed by high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS). In addition, EPHF's antioxidant capacity, acetylcholinesterase (AChE) inhibitory activity and cytotoxic activity were evaluated. EPHF was obtained by Diaion HP-20 adsorption chromatography. Phytochemical composition of EPHF was analyzed qualitatively and quantitatively using HPLC and LC-MS. Radical scavenging capacity of EPHF was estimated using 2,2-diphenyl-1-picryhydrazyl (DPPH) assay and oxygen radical absorbance capacity (ORAC) assay. The AChE inhibitory activity of EPHF was evaluated by Ellman method. Cytotoxic activity of EPHF was assessed by means of MTT assay. Eight kinds of components were identified, in which ideain with the value of 179.4 mg/g was identified to be present in the highest level in EPHF, followed by (-)-epicatechin, chlorogenic acid, cyanidin 3-arabinoside, hyperoside and isoquercitrin at the concentrations of 40.9, 10.0, 1.4, 0.4 and 0.2 mg/g, respectively. The contents of these compounds in EPHF were much higher than those in PHF and HF. In addition, EPHF exhibited strong antioxidant and AChE inhibitory activity (ORAC value: 11.65 ± 2.37 μM Trolox equivalents (TE)/mg, DPPH IC 50 value: 6.72 μg/mL, anti-AChE activity IC 50 value: 11.72 μg/mL) compared with PHF and HF. Moreover, EPHF exhibited high levels of cytotoxicity on MCF-7 and SKOV-3 human tumour cell lines in a dose-dependent manner with the IC 50 of 2.76 and 80.11 μg/mL, respectively. Macroporous resin

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.

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.

Upconversion nanoparticle-based fluorescence resonance energy transfer assay for organophosphorus pesticides.

PubMed

Long, Qian; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo

2015-06-15

This paper reports a novel nanosensor for organophosphorus pesticides based on the fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The detection mechanism is based on the facts that AuNPs quench the fluorescence of UCNPs and organophosphorus pesticides (OPs) inhibit the activity of acetylcholinesterase (AChE) which catalyzes the hydrolysis of acetylthiocholine (ATC) into thiocholine. Under the optimized conditions, the logarithm of the pesticides concentration was proportional to the inhibition efficiency. The detection limits of parathion-methyl, monocrotophos and dimethoate reached 0.67, 23, and 67 ng/L, respectively. Meanwhile, the biosensor shows good sensitivity, stability, and could be successfully applied to detection of OPs in real food samples, suggesting the biosensor has potentially extensive application clinic diagnoses assays. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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

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

Copper (II) and zinc (II) complexes with flavanone derivatives: Identification of potential cholinesterase inhibitors by on-flow assays.

PubMed

Sarria, André Lucio Franceschini; Vilela, Adriana Ferreira Lopes; Frugeri, Bárbara Mammana; Fernandes, João Batista; Carlos, Rose Maria; da Silva, Maria Fátima das Graças Fernandes; Cass, Quezia Bezerra; Cardoso, Carmen Lúcia

2016-11-01

Metal chelates strongly influence the nature and magnitude of pharmacological activities in flavonoids. In recent years, studies have shown that a promising class of flavanone-metal ion complexes can act as selective cholinesterase inhibitors (ChEIs), which has led our group to synthesize a new series of flavanone derivatives (hesperidin, hesperetin, naringin, and naringenin) complexed to either copper (II) or zinc (II) and to evaluate their potential use as selective ChEIs. Most of the synthesized complexes exhibited greater inhibitory activity against acetylcholinesterase (AChE) than against butyrylcholinesterase (BChE). Nine of these complexes constituted potent, reversible, and selective ChEIs with inhibitory potency (IC 50 ) and inhibitory constant (K i ) ranging from 0.02 to 4.5μM. Copper complexes with flavanone-bipyridine derivatives afforded the best inhibitory activity against AChE and BChE. The complex Cu(naringin)(2,2'-bipyridine) (11) gave IC 50 and K i values of 0.012±0.002 and 0.07±0.01μM for huAChE, respectively, which were lower than the inhibitory values obtained for standard galanthamine (IC 50 =206±30.0 and K i =126±18.0μM). Evaluation of the inhibitory activity of this complex against butyrylcholinesterase from human serum (huBChE) gave IC 50 and K i values of 8.0±1.4 and 2.0±0.1μM, respectively. A Liquid Chromatography-Immobilized Capillary Enzyme Reactor by UV detection (LC-ICER-UV) assay allowed us to determine the IC 50 and K i values and the type of mechanism for the best inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

Synthesis, DNA Cleavage Activity, Cytotoxicity, Acetylcholinesterase Inhibition, and Acute Murine Toxicity of Redox-Active Ruthenium(II) Polypyridyl Complexes.

PubMed

Alatrash, Nagham; Narh, Eugenia S; Yadav, Abhishek; Kim, Mahn-Jong; Janaratne, Thamara; Gabriel, James; MacDonnell, Frederick M

2017-07-06

Four mononuclear [(L-L) 2 Ru(tatpp)] 2+ and two dinuclear [(L-L) 2 Ru(tatpp)Ru(L-L) 2 ] 4+ ruthenium(II) polypyridyl complexes (RPCs) containing the 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) ligand were synthesized, in which L-L is a chelating diamine ligand such as 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me 4 phen) or 4,7-diphenyl-1,10-phenanthroline (Ph 2 phen). These Ru-tatpp analogues all undergo reduction reactions with modest reducing agents, such as glutathione (GSH), at pH 7. These, plus several structurally related but non-redox-active RPCs, were screened for DNA cleavage activity, cytotoxicity, acetylcholinesterase (AChE) inhibition, and acute mouse toxicity, and their activities were examined with respect to redox activity and lipophilicity. All of the redox-active RPCs show single-strand DNA cleavage in the presence of GSH, whereas none of the non-redox-active RPCs do. Low-micromolar cytotoxicity (IC 50 ) against malignant H358, CCL228, and MCF7 cultured cell lines was mainly restricted to the redox-active RPCs; however, they were substantially less toxic toward nonmalignant MCF10 cells. The IC 50 values for AChE inhibition in cell-free assays and the acute toxicity of RPCs in mice revealed that whereas most RPCs show potent inhibitory action against AChE (IC 50 values <15 μm), Ru-tatpp complexes as a class are surprisingly well tolerated in animals relative to other RPCs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design, synthesis and evaluation of rivastigmine and curcumin hybrids as site-activated multitarget-directed ligands for Alzheimer's disease therapy.

PubMed

Li, Yujie; Peng, Peng; Tang, Li; Hu, Yunzhen; Hu, Yongzhou; Sheng, Rong

2014-09-01

A series of novel 2-methoxy-phenyl dimethyl-carbamate derivatives were designed, synthesized and evaluated as site-activated MTDLs based on rivastigmine and curcumin. Most of them exhibited good to excellent AChE and BuChE inhibitory activities with sub-micromolar IC50 values. Among all the compounds, 6a demonstrated the most potent AChE inhibition with IC50 value of 0.097μM, which is about 20-fold than that of rivastigmine. In addition, the three selected compounds 5a, 6a and 6e demonstrated inhibitory activity against Aβ self-aggregation similar to cucurmin in TEM assay, which is obviously different from the weak activity of rivastigmine. Moreover, the hydrolysate of 6a (compound 7) also showed potent ABTS(+) scavenging and moderate copper ion chelating activity in vitro. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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.

Nicotine-Induced Airway Smooth Muscle Cell Proliferation Involves TRPC6-Dependent Calcium Influx Via α7 nAChR.

PubMed

Hong, Wei; Peng, Gongyong; Hao, Binwei; Liao, Baoling; Zhao, Zhuxiang; Zhou, Yumin; Peng, Fang; Ye, Xiuqin; Huang, Lingmei; Zheng, Mengning; Pu, Jinding; Liang, Chunxiao; Yi, Erkang; Peng, Huanhuan; Li, Bing; Ran, Pixin

2017-01-01

The proliferation of human bronchial smooth muscle cells (HBSMCs) is a key pathophysiological component of airway remodeling in chronic obstructive pulmonary disease (COPD) for which pharmacotherapy is limited, and only slight improvements in survival have been achieved in recent decades. Cigarette smoke is a well-recognized risk factor for COPD; however, the pathogenesis of cigarette smoke-induced COPD remains incompletely understood. This study aimed to investigate the mechanisms by which nicotine affects HBSMC proliferation. Cell viability was assessed with a CCK-8 assay. Proliferation was measured by cell counting and EdU immunostaining. Fluorescence calcium imaging was performed to measure intracellular Ca2+ concentration ([Ca2+]i). The results showed that nicotine promotes HBSMC proliferation, which is accompanied by elevated store-operated calcium entry (SOCE), receptor-operated calcium entry (ROCE) and basal [Ca2+]i in HBSMCs. Moreover, we also confirmed that canonical transient receptor potential protein 6 (TRPC6) and α7 nicotinic acetylcholine receptor (α7 nAChR) are involved in nicotine-induced upregulation of cell proliferation. Furthermore, we verified that activation of the PI3K/Akt signaling pathway plays a pivotal role in nicotine-enhanced proliferation and calcium influx in HBSMCs. Inhibition of α7 nAChR significantly decreased Akt phosphorylation levels, and LY294002 inhibited the protein expression levels of TRPC6. Herein, these data provide compelling evidence that calcium entry via the α7 nAChR-PI3K/Akt-TRPC6 signaling pathway plays an important role in the physiological regulation of airway smooth muscle cell proliferation, representing an important target for augmenting airway remodeling. © 2017 The Author(s). Published by 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.

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

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.

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

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.

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

DNA Methyltransferase Activity Assays: Advances and Challenges

PubMed Central

Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

2016-01-01

DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

In vitro screening for anti-cholinesterase and antioxidant activity of methanolic extracts of ayurvedic medicinal plants used for cognitive disorders.

PubMed

Mathew, Maya; Subramanian, Sarada

2014-01-01

Inhibition of Acetylcholinesterase (AChE) is still considered as the main therapeutic strategy against Alzheimer's disease (AD). Many plant derived phytochemicals have shown AChE inhibitory activity in addition to the currently approved drugs for AD. In the present study, methanolic extracts of 20 plants used in Indian Ayurvedic system of medicine for improving cognitive function were screened for acetylcholinesterase inhibitory activity by Ellman's microplate colorimetric method. Out of 20 extracts, Emblica officinalis, Nardostachys jatamansi, Nelumbo nucifera, Punica granatum and Raulfia Serpentina showed IC50 values <100 µg/ml for acetylcholinesterase inhibitory activity. Antioxidant activities of these plants were assessed by DPPH scavenging assay. Among the extracts used, antioxidant activity was highest for Terminalia chebula and Emblica officinalis with IC50 values <10 µg/ml. Considering the complex multifactorial etiology of AD, these plant extracts will be safer and better candidates for the future disease modifying therapies against this devastating disease.

Chemical Composition and Acetylcholinesterase Inhibitory Activity of Essential Oils from Piper Species.

PubMed

Xiang, Cai-Peng; Han, Jia-Xin; Li, Xing-Cong; Li, Yun-Hui; Zhang, Yi; Chen, Lin; Qu, Yan; Hao, Chao-Yun; Li, Hai-Zhou; Yang, Chong-Ren; Zhao, San-Jun; Xu, Min

2017-05-10

The essential oils (EOs) derived from aromatic plants such as Piper species are considered to play a role in alleviating neuronal ailments that are associated with inhibition of acetylcholinesterase (AChE). The chemical compositions of 23 EOs prepared from 16 Piper spp. were analyzed by both gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 76 compounds were identified in the EOs from the leaves and stems of 19 samples, while 30 compounds were detected in the EOs from the fruits of four samples. Sesquiterpenes and phenylpropanoids were found to be rich in these EOs, of which asaricin, caryophyllene, caryophyllene oxide, isospathulenol, (+)-spathulenol, and β-bisabolene are the major constituents. The EOs from the leaves and stems of Piper austrosinense, P. puberulum, P. flaviflorum, P. betle, and P. hispidimervium showed strong AChE inhibitory activity with IC 50 values in the range of 1.51 to 13.9 mg/mL. A thin-layer chromatography (TLC) bioautography assay was employed to identify active compound(s) in the most active EO from P. hispidimervium. The active compound was isolated and identified as asaricin, which gave an IC 50 value of 0.44 ± 0.02 mg/mL against AChE, comparable to galantamine with an IC 50 0.15 ± 0.01 mg/mL.

Antioxidant and acetylcholinesterase inhibitory activities in vitro of different fraction of Huperzia squarrosa (Forst.) Trevis extract and attenuation of scopolamine-induced cognitive impairment in mice.

PubMed

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

2017-02-23

Huperzia squarrosa (Forst.) Trevis is used in traditional medicine for improving memory deficits. Alkaloids, triterpenoids, flavonoids are main bioactive compounds of Huperzia squarrosa (Forst.) Trevis. This study aimed to investigate the antioxidant, AChE inhibitory activities in vitro of differents fraction of Huperzia squarrosa (Forst.) Trevis extract and neuroprotective effects of EtOAc fraction on scopolamine-induced cognitive impairment in mice. Antioxidant activity was measured by DPPH assay. AChE inhibitory effect in vitro and detail kinetic inhibition mechanism was evaluated by Ellman's assay. For in vivo assay, mice were administrated orally EtOAc fraction (150 and 300mg/kg) for fourteen days, and injected scopolamine at a dose of 1mg/kg intraperitoneally for four days to induce memory injured. The memory behaviors were evaluated using the Morris water maze. ACh levels were measured in brain tissue. Superoxide dismutase (SOD), glutathione peroxidase (GPx) activities, malondialdehyde and protein thiol groups were also evaluated in the brains. Our data also demonstrated that EtOAc fraction had the strongest antioxidant with an IC 50 value of 9.35±1.68µg/mL and AChE inhibitory activity with an IC 50 value of 23.44±3.14μg/mL in a concentration-dependent manner. Kinetic inhibition analysis indicated that EtOAc fraction was mixed inhibition type with Ki (representing the affinity of the enzyme and inhibitor) was 34.75±1.42µg/mL. Scopolamine significantly increased the escape latency time, reduced the crossings number, and swimming time in the target quadrant, while EtOAc fraction reversed these scopolamine-induced effects. EtOAc fraction significantly increased levels of acetylcholine in the brain. EtOAc fraction also significantly decreased oxidative stress in mice. Our data suggest that EtOAc fraction of Huperzia squarrosa extract exhibited a strong neuroprotective effect on cognitive impairment, and may be a potential candidate for the treatment of

New cholinesterase inhibitors for Alzheimer's disease: Structure Activity Studies (SARs) and molecular docking of isoquinolone and azepanone derivatives.

PubMed

Bacalhau, Patrícia; San Juan, Amor A; Marques, Carolina S; Peixoto, Daniela; Goth, Albertino; Guarda, Cátia; Silva, Mara; Arantes, Sílvia; Caldeira, A Teresa; Martins, Rosário; Burke, Anthony J

2016-08-01

A library of isoquinolinone and azepanone derivatives were screened for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. The strategy adopted included (a) in vitro biological assays, against eel AChE (EeAChE) and equine serum BuChE (EqBuChE) in order to determine the compounds IC50 and their dose-response activity, consolidated by (b) molecular docking studies to evaluate the docking poses and interatomic interactions in the case of the hit compounds, validated by STD-NMR studies. Compound (1f) was identified as one of these hits with an IC50 of 89.5μM for EeAChE and 153.8μM for EqBuChE, (2a) was identified as a second hit with an IC50 of 108.4μM (EeAChE) and 277.8μM (EqBuChE). In order to gain insights into the binding mode and principle active site interactions of these molecules, (R)-(1f) along with 3 other analogues (also as the R-enantiomer) were docked into both RhAChE and hBuChE models. Galantamine was used as the benchmark. The docking study was validated by performing an STD-NMR study of (1f) with EeAChE using galantamine as the benchmark. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

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.

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

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.

In Vitro Screening for Anti-Cholinesterase and Antioxidant Activity of Methanolic Extracts of Ayurvedic Medicinal Plants Used for Cognitive Disorders

PubMed Central

Mathew, Maya; Subramanian, Sarada

2014-01-01

Inhibition of Acetylcholinesterase (AChE) is still considered as the main therapeutic strategy against Alzheimer’s disease (AD). Many plant derived phytochemicals have shown AChE inhibitory activity in addition to the currently approved drugs for AD. In the present study, methanolic extracts of 20 plants used in Indian Ayurvedic system of medicine for improving cognitive function were screened for acetylcholinesterase inhibitory activity by Ellman’s microplate colorimetric method. Out of 20 extracts, Emblica officinalis, Nardostachys jatamansi, Nelumbo nucifera, Punica granatum and Raulfia Serpentina showed IC50 values <100 µg/ml for acetylcholinesterase inhibitory activity. Antioxidant activities of these plants were assessed by DPPH scavenging assay. Among the extracts used, antioxidant activity was highest for Terminalia chebula and Emblica officinalis with IC50 values <10 µg/ml. Considering the complex multifactorial etiology of AD, these plant extracts will be safer and better candidates for the future disease modifying therapies against this devastating disease. PMID:24466247

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

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.

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

A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

PubMed

O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

2017-08-01

Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1 -/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

Cellobiohydrolase (CBH) Activity Assays.

PubMed

Sharma, Hem Kanta; Qin, Wensheng; Xu, Chunbao Charles

2018-01-01

Cellulosic biomass is the most abundant biopolymer on the earth. It has great potential to quench the thirst of liquid energy by producing biofuels and thus help to mitigate human reliance on fossil fuels. Although several cellulase activity assay methods have been used to disintegrate the glycosidic bonds, the appropriate selection of substrates and synergistic involvement of multiple enzymes in hydrolytic activity is not yet fully understood. The proper quantification of hydrolytic enzymes and hydrolysates is challenging because of the heterogeneity of cellulose, changes in enzyme-substrate ratio and the presence of some inhibitory compounds like cellobiose and cellodextran. In the glycosyl hydrolase (GH) family, cellobiohydrolase (CBH) is expected to disrupt the crystalline cellulose and release the sugar molecules. Several methods have been proposed for CBH assay with slight modification in substrate and quantification of hydrolysates. However, the Avicel method is still considered as the most promising and efficient hydrolytic technique so far. The most commonly used CBH assays including Avicel and other recent methods for proper quantification are outlined in this chapter. Also a qualitative screening of CBH producing bacteria using carboxymethyl cellulose (CMC) agar plates is described.

Protection by pyridostigmine bromide of marmoset hemi-diaphragm acetylcholinesterase activity after soman exposure.

PubMed

Haigh, Julian R; Adler, Michael; Apland, James P; Deshpande, Sharad S; Barham, Charles B; Desmond, Patrick; Koplovitz, Irwin; Lenz, David E; Gordon, Richard K

2010-09-06

Pyridostigmine bromide (PB) was approved by the U.S. Food and Drug Administration (FDA) in 2003 as a pretreatment in humans against the lethal effects of the irreversible nerve agent soman (GD). Organophosphate (OP) chemical warfare agents such as GD exert their toxic effects by inhibiting acetylcholinesterase (AChE) from terminating the action of acetylcholine at postsynaptic sites in cholinergic nerve terminals (including crucial peripheral muscle such as diaphragm). As part of the post-marketing approval of PB, the FDA required (under 21CFR314, the "two animal rule") the study of a non-human primate model (the common marmoset Callithrix jacchus jacchus) to demonstrate increased survival against lethal GD poisoning, and protection of physiological hemi-diaphragm function after PB pretreatment and subsequent GD exposure. Marmosets (male and female) were placed in the following experimental groups: (i) control (saline pretreatment only), (ii) low dose PB (12.5 microg/kg), or (iii) high dose (39.5 microg/kg) PB. Thirty minutes after the PB dose, animals were challenged with either saline (control) or soman (GD, 45 microg/kg), followed 1 min later by atropine (2mg/kg) and 2-PAM (25mg/kg). After a further 16 min, animals were euthanized and the complete diaphragm removed; the right hemi-diaphragm was frozen immediately at -80 degrees C, and the left hemi-diaphragm was placed in a tissue bath for 4h (to allow for decarbamylation to occur), then frozen. AChE activities were determined using the automated WRAIR cholinesterase assay. Blood samples were collected for AChE activities prior to PB, before GD challenge, and after sacrifice. RBC-AChE was inhibited by approximately 18% and 50% at the low and high doses of PB, respectively, compared to control (baseline) activity. In the absence of PB pretreatment, the inhibition of RBC-AChE by GD was 98%. The recovery of hemi-diaphragm AChE activity after the 4h wash period (decarbamylation) was approximately 8% and 17%, at the

In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae

PubMed Central

Formagio, A.S.N.; Vieira, M.C.; Volobuff, C.R.F.; Silva, M.S.; Matos, A.I.; Cardoso, C.A.L.; Foglio, M.A.; Carvalho, J.E.

2015-01-01

The aim of this research was to investigate the antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species in vitro. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for acetylcholinesterase (AchE) inhibitors using Ellman's reagent. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI50) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 µg/mL, respectively. In addition, the Annona crassiflora and A. coriacea seed extracts were the most active among the tested extracts and the most effective against the tumor cell lines, with GI50 values below 8.90 µg/mL. The A. cacans extract displayed the lowest activity. Based on the microplate assay, the percent AchE inhibition of the extracts ranged from 12 to 52%, and the A. coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the A. crassiflora seed samples. The A. coriacea seeds contained ferulic and sinapic acid. Overall, the results indicated that A. crassiflora and A. coriacea extracts have antiproliferative and anticholinesterase properties, which opens up new possibilities for alternative pharmacotherapy drugs. PMID:25714885

In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae.

PubMed

Formagio, A S N; Vieira, M C; Volobuff, C R F; Silva, M S; Matos, A I; Cardoso, C A L; Foglio, M A; Carvalho, J E

2015-04-01

The aim of this research was to investigate the antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species in vitro. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for acetylcholinesterase (AchE) inhibitors using Ellman's reagent. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI50) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 µg/mL, respectively. In addition, the Annona crassiflora and A. coriacea seed extracts were the most active among the tested extracts and the most effective against the tumor cell lines, with GI50 values below 8.90 µg/mL. The A. cacans extract displayed the lowest activity. Based on the microplate assay, the percent AchE inhibition of the extracts ranged from 12 to 52%, and the A. coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the A. crassiflora seed samples. The A. coriacea seeds contained ferulic and sinapic acid. Overall, the results indicated that A. crassiflora and A. coriacea extracts have antiproliferative and anticholinesterase properties, which opens up new possibilities for alternative pharmacotherapy drugs.

[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.

Intraperitoneal Exposure to Nano/Microparticles of Fullerene (C60) Increases Acetylcholinesterase Activity and Lipid Peroxidation in Adult Zebrafish (Danio rerio) Brain

PubMed Central

Dal Forno, Gonzalo Ogliari; Kist, Luiza Wilges; de Azevedo, Mariana Barbieri; Fritsch, Rachel Seemann; Pereira, Talita Carneiro Brandão; Britto, Roberta Socoowski; Guterres, Sílvia Stanisçuaski; Külkamp-Guerreiro, Irene Clemes; Bonan, Carla Denise; Monserrat, José María; Bogo, Maurício Reis

2013-01-01

Even though technologies involving nano/microparticles have great potential, it is crucial to determine possible toxicity of these technological products before extensive use. Fullerenes C60 are nanomaterials with unique physicochemical and biological properties that are important for the development of many technological applications. The aim of this study was to evaluate the consequences of nonphotoexcited fullerene C60 exposure in brain acetylcholinesterase expression and activity, antioxidant responses, and oxidative damage using adult zebrafish as an animal model. None of the doses tested (7.5, 15, and 30 mg/kg) altered AChE activity, antioxidant responses, and oxidative damage when zebrafish were exposed to nonphotoexcited C60 nano/microparticles during 6 and 12 hours. However, adult zebrafish exposed to the 30 mg/kg dose for 24 hours have shown enhanced AChE activity and augmented lipid peroxidation (TBARS assays) in brain. In addition, the up-regulation of brain AChE activity was neither related to the transcriptional control (RT-qPCR analysis) nor to the direct action of nonphotoexcited C60 nano/microparticles on the protein (in vitro results) but probably involved a posttranscriptional or posttranslational modulation of this enzymatic activity. Taken together these findings provided further evidence of toxic effects on brain after C60 exposure. PMID:23865059

Phenolic composition, antioxidant and anti-acetylcholinesterase activities of the Tunisian Scabiosa arenaria.

PubMed

Besbes Hlila, Malek; Omri, Amel; Ben Jannet, Hichem; Lamari, Ali; Aouni, Mahjoub; Selmi, Boulbaba

2013-05-01

There is a need for the discovery of novel natural antioxidants and acetylcholinesterase inhibitors (AChEIs) that are safe and effective at a global level. This is the first study on antioxidant and anti-acethylcholinesterase activity of Scabiosa arenaria Forssk (Dipsacaceae). The antioxidant potential and anti-acetylcholinesterase (AChE) activity of S. arenaria were investigated. The crude, ethyl acetate (EtOAc), butanol (n-BuOH) and water extracts prepared from flowers, fruits and stems and leaves of S. arenaria were tested to determine their total polyphenol content (TPC), total flavonoid content (TFC), total condensed tannin content (CTC) and their antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), reducing power and β-carotene bleaching inhibition activity. Anti-AChE activity was also determined. EtOAc and n-BuOH fractions of fruits had both the highest (TPC) (269.09 mg gallic acid equivalents/g dry weight). The crude extract of stems and leaves had the highest TFC (10.9 mg quercetin equivalent/g dry weight). The n-BuOH fraction of stems and leaves had the highest CTC (489.75 mg catechin equivalents/g dry weight). The EtOAc fraction of flowers exhibit a higher activity in each antioxidant system with a special attention for DPPH assay (IC50 = 0.017 mg/mL) and reducing power (EC50 = 0.02 mg/mL). The EtOAc and n-BuOH fractions of stems and leaves showed strong inhibition of AChE (IC50 = 0.016 and 0.029 mg/mL, respectively). These results suggest the potential of S. arenaria as a possible source of novel compounds and as an alternative antioxidant and AChEIs.

Endoglucanase (EG) Activity Assays.

PubMed

Rahman, M Shafiqur; Fernando, Sheran; Ross, Brian; Wu, Jiangning; Qin, Wensheng

2018-01-01

Cellulosic biomass, the most common organic compound of primary energy source on earth, is a network of interwoven biopolymers of plant cell walls. Degradation of cellulose is important for global carbon recycling. Moreover, biofuel, a renewable fuel whose energy can be derived from cellulosic biomass by enzymatic hydrolysis of cellulases. Among cellulases are endoglucases that act synergistically for subsequent hydrolytic reactions to break down the polymeric cellulose. However, in cellulolytic enzyme activity endoglucanase plays a prominent role in initiating and sustaining the hydrolytic process. Endoglucanase randomly cleaves the cellulose polymer into smaller sugar and oligomeric polysaccharides. Characterization and quantification of endoglucanase activity is important for industry and in the overall study of cellulose degradation. All assays including those for endoglucanase fall into two broad categories either qualitative or quantitative. Quantitative assays can tell if the enzyme is present, how much and its activity. Measurement can be done indirectly using a secondary colorimetric product like (1) molybdenum blue, (2) 3-amino-5-nitrosalicylic acid, (3) bicinchoninic acid (BCA), and (4) 2-cyanoacetamide or directly using an antibody in an ELISA. In this chapter, we discuss several common protocols for the measurement of endoglucanase activity.

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

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

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.

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

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.

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

PubMed

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

2013-03-01

Patients diagnosed with Alzheimer's disease (AD) show a characteristic neurochemical deficit of acetylcholine, especially in the basal forebrains. The use of acetylcholinesterase (AChE) inhibitors to retard the hydrolysis of acetylcholine has been suggested as a promising strategy for AD treatment. In this study, we evaluated the acetylcholinesterase inhibitory (AChEI) activities of 134 extracts obtained from 45 species of marine sponges. Thin-layer chromatography (TLC) and microplate assays reveal potent acetylcholinsterase inhibitory activities of two AcOEt extracts from the sponges Pericharax heteroraphis and Amphimedon navalis PULITZER-FINALI. We further investigated the inhibitory kinetics of the extracts and found them to display mixed competitive/noncompetitive inhibition and associated their inhibitory activity partly to terpenoids. Acetylcholinesterase inhibitors from marine organisms have been rarely studied, and this study demonstrated the potential of marine sponges as a source of pharmaceutical leads against neurodegenerative diseases. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

Antioxidant, anticholinesterase and antibacterial activities of Stachys guyoniana and Mentha aquatica.

PubMed

Ferhat, Maria; Erol, Ebru; Beladjila, Khadidja Aya; Çetintaş, Yunus; Duru, Mehmet Emin; Öztürk, Mehmet; Kabouche, Ahmed; Kabouche, Zahia

2017-12-01

Stachys guyoniana Noë ex. Batt. and Mentha aquatica L. are two Algerian Lamiaceae used in folk medicine. To investigate their antioxidant, anticholinesterase and antibacterial activities. n-Butanol (BESG), ethyl acetate (EESG) and chloroform (CESG) extracts of S. guyoniana and methanol (MEMA) and chloroform (CEMA) aerial part extracts of M. aquatica and methanol (MERMA) and acetone (AERMA) roots extracts of M. aquatica were evaluated for their antioxidant activity by the β-carotene-linoleic acid, DPPH • and ABTS • +  scavenging, CUPRAC and metal chelating assays. The anticholinesterase activity was tested against AChE and BChE. The antibacterial activity was assessed by MICs determination against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella heidelberg, Klebsiella pneumoniae, Enterobacter aerogenes and Morganella morganii strains. In the β-carotene test, the CESG (IC 50 : 2.3 ± 1.27 μg/mL) exhibited the highest activity. The BESG was the best scavenger of DPPH • (IC 50 : 2.91 ± 0.14 μg/mL). In the ABTS test, AERMA was the most active (IC 50 : 4.21 ± 0.28 μg/mL). However, with the CUPRAC, the BESG exhibited the best activity (A 0.50 : 0.15 ± 0.05 μg/mL) and was active in metal chelating assay with 48% inhibition at 100 μg/mL. The BESG was the best AChE inhibitor (IC 50 : 5.78 ± 0.01 μg/mL) however, the AERMA showed the highest BChE inhibitory activity (IC 50 : 19.23 ± 1.42 μg/mL). The tested extracts exhibited a good antibacterial activity. This study demonstrated good antioxidant, anticholinesterase and antibacterial potential of S. guyoniana and M. aquatica, which fits in well with their use in folk medicine.

In Search of an Effective in vivo Reactivator for Organophosphorus Nerve Agent-Inhibited Acetylcholinesterase in the Central Nervous System

DTIC Science & Technology

2012-01-01

monoisonitrosoacetone (MINA) crossed BBB, provided some degree of CNS AChE reactivation, enhanced survival, and mitigated the seizure activity following nerve agent...tissues (brain regions, diaphragm, heart, skeletal muscle) were collected. AChE activity was measured using the Ellman assay. In GB exposure, pro...therapy. Protecting and/or restoring AChE activity in the brain is a major goal in the treatment of nerve agent intoxication. Our long-term goal is to

Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery.

PubMed

González-Cestari, Tatiana F; Henderson, Brandon J; Pavlovicz, Ryan E; McKay, Susan B; El-Hajj, Raed A; Pulipaka, Aravinda B; Orac, Crina M; Reed, Damon D; Boyd, R Thomas; Zhu, Michael X; Li, Chenglong; Bergmeier, Stephen C; McKay, Dennis B

2009-02-01

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

Effect of Novel Negative Allosteric Modulators of Neuronal Nicotinic Receptors on Cells Expressing Native and Recombinant Nicotinic Receptors: Implications for Drug Discovery

PubMed Central

González-Cestari, Tatiana F.; Henderson, Brandon J.; Pavlovicz, Ryan E.; McKay, Susan B.; El-Hajj, Raed A.; Pulipaka, Aravinda B.; Orac, Crina M.; Reed, Damon D.; Boyd, R. Thomas; Zhu, Michael X.; Li, Chenglong; Bergmeier, Stephen C.; McKay, Dennis B.

2009-01-01

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of α3β4* nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant α3β4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native α3β4* nAChR, with IC50 values ranging from 0.4 to 13.0 μM. Using cells expressing recombinant α3β4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC50 values ranging from 0.7 to 38.2 μM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 μM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery. PMID:18984653

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

PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system.

PubMed

Rigo, Flavia Karine; Rossato, Mateus Fortes; Trevisan, Gabriela; De Prá, Samira Dal-Toé; Ineu, Rafael Porto; Duarte, Mariane Bernardo; de Castro Junior, Célio José; Ferreira, Juliano; Gomez, Marcus Vinicius

2017-10-01

Cholinergic agents cause antinociception by mimicking the release of acetylcholine (ACh) from spinal cholinergic nerves. PhKv is a peptide isolated from the venom of the armed spider Phoneutria nigriventer. It has an antiarrythmogenic activity that involves the enhanced release of acetylcholine. The aim of this study was to investigate whether PhKv had an antinociceptive action in mice. Male albino Swiss mice (25-35g) were used in this study. The PhKv toxin was purified from a PhTx3 fraction of the Phoneutria nigriventer spider's venom. Because of its peptide nature, PhKv is not orally available and it was delivered directly into the central nervous system by an intrathecal (i.t.) route. PhKV on the thermal and mechanical sensitivity was evaluated using plantar test apparatus and the up-and-down method. The analgesic effects of PhKv were studied in neuropathic pain (CCI) and in the peripheral capsicin test. In order to test whether PhKv interfered with the cholinergic system, the mice were pre-treated with atropine (5mg/kg, i.p.) or mecamylamine (0.001mg/kg, i.p.) and the PhKv toxin (30pmol/site i.t.) or neostigmine (100pmol/site) were applied 15min before the intraplantar capsaicin (1nmol/paw) administrations. To investigate PhKv action on the AChE activities, was performed in vitro and ex vivo assay for AChE. For the in vitro experiments, mice spinal cord supernatants of tissue homogenates (1mg/ml) were used as source of AChE activity. The AChE assay was monitored at 37°C for 10min in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 405nm. PhKv (30 and 100pmol/site, i.t.) had no effect on the thermal or mechanical sensitivity thresholds. However, in a chronic constriction injury model of pain, PhKv (10pmol/site, i.t.) caused a robust reduction in mechanical withdrawal with an antinociceptive effect that lasted 4h. A pretreatment in mice with PhKv (30pmol/site, i.t.) or neostigmine (100pmol/site, i.t.) 15min before an intraplantar injection of

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.

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.

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.

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

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

Inflammation triggers constitutive activity and agonist-induced negative responses at M(3) muscarinic receptor in dental pulp.

PubMed

Sterin-Borda, Leonor; Orman, Betina; De Couto Pita, Alejandra; Borda, Enri

2011-02-01

The purpose of this study was to investigate whether the inflammation of rat dental pulp induces the muscarinic acetylcholine receptor (mAChR) constitutive receptor activity. Pulpitis was induced with bacterial lipolysaccharide in rat incisors dental pulp. Saturation assay with [(3)H]-quinuclidinyl benzilate ([(3)H] QNB), competitive binding with different mAChR antagonist subtypes, and nitric oxide synthase (NOS) activity were performed. A drastic change in expression and response to mAChR subtypes was observed in pulpitis. Inflamed pulp expressed high number of M(3) mAChR of high affinity, whereas the M(1) mAChR is the main subtype displayed in normal pulp. Consistent with the identification of the affinity constant (Ki) of M(3) and Ki of M(1) in both pulpitis and in normal pulps are the differences in the subtype functionality of these cells. In pulpitis, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) exerted an inhibitory action on NOS activity that was blocked by J 104129 fumarate (highest selective affinity to M(3) mAChR). In normal pulps, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) has no effect. NOS basal activity was 5.9 times as high in pulpitis as in the normal pulp as a result of the activation of inducible NOS. The irreversible pulpitis could induce a mAChR alteration, increasing the high-affinity receptor density and transduction-coupling efficiency of inducible NOS activity, leading to a spontaneously active conformation of the receptor. Pilocarpine acting as an inverse agonist might be useful therapeutically to prevent necrosis and subsequent loss of dental pulp. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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.

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.

Biochemical assays on plasminogen activators and hormones from kidney sources

NASA Technical Reports Server (NTRS)

Barlow, Grant H.; Lewis, Marian L.; Morrison, Dennis R.

1988-01-01

Investigations were established for the purpose of analyzing the conditioned media from human embryonic kidney cell subpopulations separated in space by electrophoresis. This data is based on the experiments performed on STS-8 on the continuous flow electrophoresis system. The primary biological activity that was analyzed was plasminogen activator activity, but some assays for erythropoeitin and human granulocyte colony stimulating activity were also performed. It is concluded that a battery of assays are required to completely define the plasminogen activator profile of a conditioned media from cell culture. Each type of assay measures different parts of the mixture and are influenced by different parameters. The functional role of each assay is given along with an indication of which combination of assays are required to answer specific questions. With this type of information it is possible by combinations of assays with mathematical analysis to pinpoint a specific component of the system.

Different Cholinesterase Inhibitor Effects on CSF Cholinesterases in Alzheimer Patients

PubMed Central

Nordberg, Agneta; Darreh-Shori, Taher; Peskind, Elaine; Soininen, Hilkka; Mousavi, Malahat; Eagle, Gina; Lane, Roger

2014-01-01

Background The current study aimed to compare the effects of different cholinesterase inhibitors on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and protein levels, in the cerebrospinal fluid (CSF) of Alzheimer disease (AD) patients. Methods and Findings AD patients aged 50–85 years were randomized to open-label treatment with oral rivastigmine, donepezil or galantamine for 13 weeks. AChE and BuChE activities were assayed by Ellman’s colorimetric method. Protein levels were assessed by enzyme-linked immunosorbent assay (ELISA). Primary analyses were based on the Completer population (randomized patients who completed Week 13 assessments). 63 patients were randomized to treatment. Rivastigmine was associated with decreased AChE activity by 42.6% and decreased AChE protein levels by 9.3%, and decreased BuChE activity by 45.6% and decreased BuChE protein levels by 21.8%. Galantamine decreased AChE activity by 2.1% and BuChE activity by 0.5%, but increased AChE protein levels by 51.2% and BuChE protein levels by10.5%. Donepezil increased AChE and BuChE activities by 11.8% and 2.8%, respectively. Donepezil caused a 215.2%increase in AChE and 0.4% increase in BuChE protein levels. Changes in mean AChE-Readthrough/Synaptic ratios, which might reflect underlying neurodegenerative processes, were 1.4, 0.6, and 0.4 for rivastigmine, donepezil and galantamine, respectively. Conclusion The findings suggest pharmacologically-induced differences between rivastigmine, donepezil and galantamine. Rivastigmine provides sustained inhibition of AChE and BuChE, while donepezil and galantamine do not inhibit BuChE and are associated with increases in CSF AChE protein levels. The clinical implications require evaluation. PMID:19199870

Different cholinesterase inhibitor effects on CSF cholinesterases in Alzheimer patients.

PubMed

Nordberg, Agneta; Darreh-Shori, Taher; Peskind, Elaine; Soininen, Hilkka; Mousavi, Malahat; Eagle, Gina; Lane, Roger

2009-02-01

The current study aimed to compare the effects of different cholinesterase inhibitors on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and protein levels, in the cerebrospinal fluid (CSF) of Alzheimer disease (AD) patients. AD patients aged 50-85 years were randomized to open-label treatment with oral rivastigmine, donepezil or galantamine for 13 weeks. AChE and BuChE activities were assayed by Ellman's colorimetric method. Protein levels were assessed by enzyme-linked immunosorbent assay (ELISA). Primary analyses were based on the Completer population (randomized patients who completed Week 13 assessments). 63 patients were randomized to treatment. Rivastigmine was associated with decreased AChE activity by 42.6% and decreased AChE protein levels by 9.3%, and decreased BuChE activity by 45.6% and decreased BuChE protein levels by 21.8%. Galantamine decreased AChE activity by 2.1% and BuChE activity by 0.5%, but increased AChE protein levels by 51.2% and BuChE protein levels by 10.5%. Donepezil increased AChE and BuChE activities by 11.8% and 2.8%, respectively. Donepezil caused a 215.2% increase in AChE and 0.4% increase in BuChE protein levels. Changes in mean AChE-Readthrough/Synaptic ratios, which might reflect underlying neurodegenerative processes, were 1.4, 0.6, and 0.4 for rivastigmine, donepezil and galantamine, respectively. The findings suggest pharmacologically-induced differences between rivastigmine, donepezil and galantamine. Rivastigmine provides sustained inhibition of AChE and BuChE, while donepezil and galantamine do not inhibit BuChE and are associated with increases in CSF AChE protein levels. The clinical implications require evaluation.

A novel clot lysis assay for recombinant plasminogen activator.

PubMed

Jamialahmadi, Oveis; Fazeli, Ahmad; Hashemi-Najafabadi, Sameereh; Fazeli, Mohammad Reza

2015-03-01

Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

Biological activity of Myrtaceae plant essential oils and their major components against Drosophila suzukii (Diptera: Drosophilidae).

PubMed

Jang, Miyeon; Kim, Junheon; Yoon, Kyungjae Andrew; Lee, Si Hyeock; Park, Chung Gyoo

2017-02-01

The spotted-wing drosophila (SWD), Drosophila suzukii (Matsumura), is a globally invasive and serious pest of numerous soft-skinned fruit crops. Assessments were made of fumigant and contact toxicities of 12 Myrtaceae plant essential oils (EOs) and their components. For determining the mode of action of major components of active EOs, their activities against acetylcholinesterase (AChE) and Glutathione S-transferase (GST) were also assessed. Strong fumigant and contact toxicities were observed from EOs of Eucalyptus citriodora and Melaleuca teretifolia. The main components of E. citriodora were citronellal and isopulegol, whereas those of M. teretifolia were neral and geranial. Geranial showed the strongest fumigant activity, followed by citronellal or neral, M. teretifolia EO, isopulegol and E. citriodora EO. In contact toxicity assays, geranial also exhibited the strongest insecticidal activity, followed by neral or M. teretifolia EO, citronellol, citronellal, isopulegol and E. citriodora EO. Among the major components, all compounds showed low AChE inhibitory activity, while neral and geranial showed GST inhibitory activity against SWD. Myrtaceae plant EOs and their components have an excellent potential for being used in the control of SWD adults and could be useful in the development of more effective natural compounds as alternatives to synthetic pesticides. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

PubMed

Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

2013-01-01

HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

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 Novel Direct Factor Xa Inhibitory Peptide with Anti-Platelet Aggregation Activity from Agkistrodon acutus Venom Hydrolysates.

PubMed

Chen, Meimei; Ye, Xiaohui; Ming, Xin; Chen, Yahui; Wang, Ying; Su, Xingli; Su, Wen; Kong, Yi

2015-06-02

Snake venom is a natural substance that contains numerous bioactive proteins and peptides, nearly all of which have been identified over the last several decades. In this study, we subjected snake venom to enzymatic hydrolysis to identify previously unreported bioactive peptides. The novel peptide ACH-11 with the sequence LTFPRIVFVLG was identified with both FXa inhibition and anti-platelet aggregation activities. ACH-11 inhibited the catalytic function of FXa towards its substrate S-2222 via a mixed model with a Ki value of 9.02 μM and inhibited platelet aggregation induced by ADP and U46619 in a dose-dependent manner. Furthermore, ACH-11 exhibited potent antithrombotic activity in vivo. It reduced paralysis and death in an acute pulmonary thrombosis model by 90% and attenuated thrombosis weight in an arterio-venous shunt thrombosis model by 57.91%, both at a dose of 3 mg/kg. Additionally, a tail cutting bleeding time assay revealed that ACH-11 did not prolong bleeding time in mice at a dose of 3 mg/kg. Together, our results reveal that ACH-11 is a novel antithrombotic peptide exhibiting both FXa inhibition and anti-platelet aggregation activities, with a low bleeding risk. We believe that it could be a candidate or lead compound for new antithrombotic drug development.

A microtitre plate assay for measuring glycosidase activity.

PubMed

Ball, Andrea L; Chambers, Kirsty A; Hewinson, Meera; Navaratnarajah, Sambavi; Samrin, Lamia; Thomas, Nesta; Tyler, Abigail E H; Wall, Amanda J; Lloyd, Matthew D

2008-02-01

Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC(50) and K(i) values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors.

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

Oral administration of pyridostigmine bromide and huperzine A protects human whole blood cholinesterases from ex vivo exposure to soman.

PubMed

Gordon, Richard K; Haigh, Julian R; Garcia, Gregory E; Feaster, Shawn R; Riel, Michael A; Lenz, David E; Aisen, Paul S; Doctor, Bhupendra P

2005-12-15

Cholinesterases (ChEs) are classified as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selected inhibitors. The activities of AChE in red blood cells (RBC-AChE) and BChE in serum can be used as potential biomarkers of suppressed and/or heightened activity in the central and peripheral nervous systems. Exposure to organophosphate (OP) chemical warfare agents (CWAs), pesticides, anesthetics, and a variety of drugs such as cocaine, as well as some neurodegenerative and liver disease states, selectively reduces AChE or BChE activity. In humans, the toxicity of pesticides is well documented. Therefore, blood cholinesterase activity can be exploited as a tool for confirming exposure to these agents and possible treatments. Current assays for measurement of RBC-AChE and serum BChE require several labor-intensive processing steps, suffer from wide statistical variation, and there is no inter-laboratory conversion between methods. These methods, which determine only the serum BChE or RBC-AChE but not both, include the Ellman, radiometric, and deltapH (modified Michel) methods. In contrast, the Walter Reed Army Institute of Research Whole Blood (WRAIR WB, US Patent #6,746,850) cholinesterase assay rapidly determines the activity of both AChE and BChE in unprocessed (uncentrifuged) whole blood, uses a minimally invasive blood sampling technique (e.g., blood from a finger prick), and is semi-automated for high-throughput using the Biomek 2000 robotic system. To date, the WRAIR whole blood assay was used to measure AChE and BChE activities in human blood from volunteers in FDA clinical trials. In the first FDA study, 24 human subjects were given either 30 mg PB orally (n = 19) or placebo (n = 5). Blood samples were obtained pre-dosing and 2.5, 5, 8, and 24 h post-dosing. The samples were analyzed for AChE and BChE activity using the WRAIR WB robotic system, and for PB concentration by HPLC. We found that

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.

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).

Chemical composition, antioxidant, anticholinesterase, antimicrobial and antibiofilm activities of essential oil and methanolic extract of Anthemis stiparum subsp. sabulicola (Pomel) Oberpr.

PubMed

Chemsa, Ahmed Elkhalifa; Zellagui, Amar; Öztürk, Mehmet; Erol, Ebru; Ceylan, Ozgür; Duru, Mehmet Emin; Lahouel, Mesbah

2018-06-01

Anthemis species are traditionally used to treat infectious and inflammatory processes, among others clinical disturbances. In the current study, the chemical composition, the total phenolic and flavonoid contents, the antioxidant, anticholinesterase, antimicrobial, and antibiofilm activities of Anthemis stiparum subsp. sabulicola aerial parts methanolic extract (As-ME) and essential oil (As-EO) were investigated. The chemical composition of As-EO was established by GC-MS and GC-FID. Total phenolic and flavonoid contents of As-ME were spectrophotometrically determined. Diphenyl-1-picrylhydrazyl (DPPH ● ) radical scavenging, cupric reducing antioxidant capacity (CUPRAC) and β-carotene bleaching assays were applied to evaluate the antioxidant potential. The anticholinesterase activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes were carried out spectrophotometrically. The antimicrobial activity was assessed by Minimal Inhibitory Concentration (MIC) using broth microdilution method against 7 ATCC ® bacterial and one ATCC ® yeast reference strains. The antibiofilm effect was determined quantifying the percentage of adhesion inhibition. GC-MS and GC-FID identified 72 compounds (99.02%), being As-EO predominantly constituted by germacrene D (11.13%), t-cadinol (11.01%), camphor (6.73%), spathulenol (6.50%) and isoamyl salicylate (6.45%). The total phenolic and flavonoid contents of As-ME were 13.6 ± 0.03 and 5.9 ± 0.04 pyrocatechol equivalents and quercetin equivalents, respectively. In β-carotene-linoleic acid assay, As-ME showed the best lipid peroxidation inhibition activity with an IC 50  = 9.96 μg/mL followed by As-EO with an IC 50  = 619.98 μg/mL. In contrast, in DPPH assay, As-ME and As-EO showed moderate to low activity with an IC 50  = 92.69 μg/mL for As-ME and 917.69 μg/mL for As-EO. While in CUPRAC assay, As-EO and As-ME indicated a less to moderate reducing activity. As-ME inhibited AChE

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.

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.

RNA Cap Methyltransferase Activity Assay

PubMed Central

Trotman, Jackson B.; Schoenberg, Daniel R.

2018-01-01

Methyltransferases that methylate the guanine-N7 position of the mRNA 5′ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments. PMID:29644259

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

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.

Phenolic, flavonoid contents, anticholinesterase and antioxidant evaluation of Iris germanica var; florentina.

PubMed

Ullah, Farhat; Ayaz, Muhammad; Sadiq, Abdul; Hussain, Abid; Ahmad, Sajjad; Imran, Muhammad; Zeb, Anwar

2016-06-01

This study was designed to investigate antioxidant and anticholinesterase potential of Iris germanica var; florentina. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potential of plant samples were investigated by Ellman's assay. Antioxidant activity was performed using DPPH, H2O2 and ABTS free radical scavenging assays. Total phenolics and flavonoids contents were expressed in mg GAE/g dry weight and mg RTE/g, respectively. In AChE inhibition assay, Ig.Fl, Ig.Sp and Ig.Cf fractions exhibited highest activity with IC50 values of < 0.1, 5.64 and 19 μg/mL, respectively. In BChE inhibitory assay, Ig.Fl, Ig.Sp, Ig.Cf and Ig.Cr were most active with IC50 of < 0.1, < 0.1, 31 and 78 μg/mL, respectively. In DPPH assay, Ig.Fl and Ig.Cf exhibited highest inhibition of free radicals, 80.52% (IC50 = 9 μg/mL) and 78.30% (IC50 = 8 μg/mL), respectively. In ABTS assay Ig.Cr, Ig.Cf, Ig.Fl and Ig.Sp exhibited IC50 values of < 0.1, 2, 2 and 3 μg/mL, respectively.

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.

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.

A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

ERIC Educational Resources Information Center

Harding, Ethelynda E.; Kimsey, R. Scott

1998-01-01

Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

Use of a novel radiometric method to assess the inhibitory effect of donepezil on acetylcholinesterase activity in minimally diluted tissue samples

PubMed Central

Kikuchi, Tatsuya; Okamura, Toshimitsu; Arai, Takuya; Obata, Takayuki; Fukushi, Kiyoshi; Irie, Toshiaki; Shiraishi, Tetsuya

2010-01-01

Background and purpose: Cholinesterase inhibitors have been widely used for the treatment of patients with dementia. Monitoring of the cholinesterase activity in the blood is used as an indicator of the effect of the cholinesterase inhibitors in the brain. The selective measurement of cholinesterase with low tissue dilution is preferred for accurate monitoring; however, the methods have not been established. Here, we investigated the effect of tissue dilution on the action of cholinesterase inhibitors using a novel radiometric method with selective substrates, N-[14C]methylpiperidin-4-yl acetate ([14C]MP4A) and (R)-N-[14C]methylpiperidin-3-yl butyrate ([14C]MP3B_R), for AChE and butyrylcholinesterase (BChE) respectively. Experimental approach: We investigated the kinetics of hydrolysis of [14C]-MP4A and [14C]-MP3B_R by cholinesterases, and evaluated the selectivity of [14C]MP4A and [14C]MP3B_R for human AChE and BChE, respectively, compared with traditional substrates. Then, IC50 values of cholinesterase inhibitors in minimally diluted and highly diluted tissues were measured with [14C]MP4A and [14C]MP3B_R. Key results: AChE and BChE activities were selectively measured as the first-order hydrolysis rates of [14C]-MP4A and [14C]MP3B_R respectively. The AChE selectivity of [14C]MP4A was an order of magnitude higher than traditional substrates used for the AChE assay. The IC50 values of specific AChE and BChE inhibitors, donepezil and ethopropazine, in 1.2-fold diluted human whole blood were much higher than those in 120-fold diluted blood. In addition, the IC50 values of donepezil in monkey brain were dramatically decreased as the tissue was diluted. Conclusions and implications: This method would effectively monitor the activity of cholinesterase inhibitors used for therapeutics, pesticides and chemical warfare agents. PMID:20401964

Selectivity of coronaridine congeners at nicotinic acetylcholine receptors and inhibitory activity on mouse medial habenula.

PubMed

Arias, Hugo R; Jin, Xiaotao; Feuerbach, Dominik; Drenan, Ryan M

2017-11-01

The inhibitory activity of coronaridine congeners on human (h) α4β2 and α7 nicotinic acetylcholine receptors (AChRs) is determined by Ca 2+ influx assays, whereas their effects on neurons in the ventral inferior (VI) aspect of the mouse medial habenula (MHb) are determined by patch-clamp recordings. The Ca 2+ influx results clearly establish that coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to hα4β2 and hα7 subtypes, and with the following potency sequence, for hα4β2: (±)-18-methoxycoronaridine [(±)-18-MC]>(+)-catharanthine>(±)-18-methylaminocoronaridine [(±)-18-MAC] ∼ (±)-18-hydroxycoronaridine [(±)-18-HC]; and for hα7: (+)-catharanthine>(±)-18-MC>(±)-18-HC>(±)-18-MAC. Interestingly, the inhibitory potency of (+)-catharanthine (27±4μM) and (±)-18-MC (28±6μM) on MHb (VI) neurons was lower than that observed on hα3β4 AChRs, suggesting that these compounds inhibit a variety of endogenous α3β4* AChRs. In addition, the interaction of bupropion with (-)-ibogaine sites on hα3β4 AChRs is tested by [ 3 H]ibogaine competition binding experiments. The results indicate that bupropion binds to ibogaine sites at desensitized hα3β4 AChRs with 2-fold higher affinity than at resting receptors, suggesting that these compounds share the same binding sites. In conclusion, coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to other AChRs, by interacting with the bupropion (luminal) site. Coronaridine congeners also inhibit α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets for their anti-addictive activities. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selectivity of coronaridine congeners at nicotinic acetylcholine receptors and inhibitory activity on mouse medial habenula

PubMed Central

Arias, Hugo R.; Jin, Xiaotao; Feuerbach, Dominik; Drenan, Ryan M.

2018-01-01

The inhibitory activity of coronaridine congeners on human (h) α4β2 and α7 nicotinic acetylcholine receptors (AChRs) is determined by Ca2+ influx assays, whereas their effects on neurons in the ventral inferior (VI) aspect of the mouse medial habenula (MHb) are determined by patch-clamp recordings. The Ca2+ influx results clearly establish that coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to hα4β2 and hα7 subtypes, and with the following potency sequence, for hα4β2: (±)-18-methoxycoronaridine [(±)-18-MC] > (+)-catharanthine > (±)-18-methylaminocoronaridine [(±)-18-MAC] ∼ (±)-18-hydroxycoronaridine [(±)-18-HC]; and for hα7: (+)-catharanthine > (±)-18-MC > (±)-18-HC > (±)-18-MAC. Interestingly, the inhibitory potency of (+)-catharanthine (27 ± 4 μM) and (±)-18-MC (28 ± 6 μM) on MHb (VI) neurons was lower than that observed on hα3β4 AChRs, suggesting that these compounds inhibit a variety of endogenous α3β4* AChRs. In addition, the interaction of bupropion with (−)-ibogaine sites on hα3β4 AChRs is tested by [3H]ibogaine competition binding experiments. The results indicate that bupropion binds to ibogaine sites at desensitized hα3β4 AChRs with 2-fold higher affinity than at resting receptors, suggesting that these compounds share the same binding sites. In conclusion, coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to other AChRs, by interacting with the bupropion (luminal) site. Coronaridine congeners also inhibit α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets for their anti-addictive activities. PMID:29042244

Active Cdk5 Immunoprecipitation and Kinase Assay

PubMed Central

Bankston, Andrew N.; Ku, Li; Feng, Yue

2017-01-01

Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal amounts of total protein between experimental groups. Washes are then performed to remove extraneous proteins and equilibrate the Cdk5-activator complexes in the kinase buffer. Cdk5 is then incubated with histone H1, a well-established in vitro target of Cdk5, and [γ-32P]ATP. Reactions are resolved by SDS-PAGE and transferred to membranes for visualization of H1 phosphorylation and immunoblot of immunoprecipitated Cdk5 levels. We have used this assay to establish p39 as the primary activator for Cdk5 in the oligodendroglial lineage. However, this assay is amenable to other cell lineages or tissues with appropriate adjustments made to lysis conditions. PMID:28868329

Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors

USGS Publications Warehouse

Rubio, Fernando; Kamp, Lisa; Carpino, Justin; Faltin, Erin; Loftin, Keith A.; Molgó, Jordi; Aráoz, Rómulo

2014-01-01

Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

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.

Antioxidant activity, acetylcholinesterase inhibitory potential and phytochemical analysis of Sarcocephalus latifolius Sm. bark used in traditional medicine in Sudan.

PubMed

Osama, Alsiddig; Awadelkarim, Sufyan; Ali, Amna

2017-05-18

Sarcocephalus latifolius is used as a traditional medicine for curing many diseases in Sudan. The main objective of the current study was to determine the antioxidant activity and acetylcholinesterase inhibition (AChEI) of S. latifolius, and to estimate its total phenolic and flavonoid contents. Antioxidant activity of the tested plant extracts was carried out by determining their ability to scavenge the 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical. On the other hand, AChE inhibitory activity was determined spectrophotometrically using the Ellman's colorimetric method. The levels of total phenols and flavonoids were determined quantitatively using spectrophotometric methods. MTT assay was consumed to assess the cytotoxic effect of the most active fractions. These fractions were subjected to phytochemical analysis using GC-MS techniques to determine thier chemical composition. Hexane and chloroform fractions exhibited the highest antioxidant activity with IC 50 values of (0.098 ± 0.08 and 0.099 ± 0.029 mg/ml) respectively. Standard propyl gallate had the lowest IC 50 value of 0.0414 ± 0.11 mg/ml. The ethanolic crude extract showed low AChEI activity with 40.2 ± 0.10%. High concentrations of phenolic and flavonoid contents were observed. GCMS revealed the presence of well-known antioxidants compounds e.g. Vitamin E and caffeic acid. The ethanolic extract of bark of S. latifolius showed potent antioxidant effects and low AChEI activity, high phenolic and flavonoid contents and presence of pharmacologically active compounds. These findings explain its wide usages in traditional medicine.

Ramalina capitata (Ach.) Nyl. acetone extract: HPLC analysis, genotoxicity, cholinesterase, antioxidant and antibacterial activity.

PubMed

Zrnzevic, Ivana; Stankovic, Miroslava; Stankov Jovanovic, Vesna; Mitic, Violeta; Dordevic, Aleksandra; Zlatanovic, Ivana; Stojanovic, Gordana

2017-01-01

In the present investigation, effects of Ramalina capitata acetone extract on micronucleus distribution on human lymphocytes, on cholinesterase activity and antioxidant activity (by the CUPRAC method) were examined, for the first time as well as its HPLC profile. Additionally, total phenolic compounds (TPC), antioxidant properties (estimated via DPPH, ABTS and TRP assays) and antibacterial activity were determined. The predominant phenolic compounds in this extract were evernic, everninic and obtusatic acids. Acetone extract of R. capitata at concentration of 2 μg mL -1 decreased a frequency of micronuclei (MN) for 14.8 %. The extract reduces the concentration of DPPH and ABTS radicals for 21.2 and 36.1 % (respectively). Values for total reducing power (TRP) and cupric reducing capacity (CUPRAC) were 0.4624 ± 0.1064 μg ascorbic acid equivalents (AAE) per mg of dry extract, and 6.1176 ± 0.2964 μg Trolox equivalents (TE) per mg of dry extract, respectively. The total phenol content was 670.6376 ± 66.554 μg galic acid equivalents (GAE) per mg of dry extract. Tested extract at concentration of 2 mg mL -1 exhibited inhibition effect (5.2 %) on pooled human serum cholinesterase. The antimicrobial assay showed that acetone extract had inhibition effect towards Gram-positive strains. The results of manifested antioxidant activity, reducing the number of micronuclei in human lymphocytes, and antibacterial activity recommends R. capitata extract for further in vivo studies.

Biological Activities and Composition of Ferulago carduchorum Essential Oil

PubMed Central

Golfakhrabadi, Fereshteh; Khanavi, Mahnaz; Ostad, Seyed Nasser; Saeidnia, Soodabeh; Vatandoost, Hassan; Abai, Mohammad Reza; Hafizi, Mitra; Yousefbeyk, Fatemeh; Rad, Yaghoob Razzaghi; Baghenegadian, Ameneh; Ardekani, Mohammad Reza Shams

2015-01-01

Background: Ferulago carduchorum Boiss and Hausskn belongs to the Apiaceae family. This plant grows in west part of Iran that local people added it to dairy and oil ghee to delay expiration date and give them a pleasant taste. The aim of this study was to investigate the antioxidant, antimicrobial, acetyl cholinesterase inhibition, cytotoxic, larvicidal activities and composition of essential oil of F. carduchorum. Methods: Acetyl cholinesterase (AChE) inhibitory, larvicidal activities and chemical composition of essential oil of F. carduchorum were investigated. Besides, antioxidant, antimicrobial and cytotoxic activities of essential oil were tested using DPPH, microdilution method and MTT assay, respectively. Results: The major components of essential oil were (z)-β-ocimene (43.3%), α-pinene (18.23%) and bornyl acetate (3.98%). Among 43 identified components, monoterpenes were the most compounds (84.63%). The essential oil had noticeable efficiency against Candida albicans (MIC= 2340 μg ml−1) and it was effective against Anopheles stephensi with LC50 and LC90 values of 12.78 and 47.43 ppm, respectively. The essential oil could inhibit AChE (IC50= 23.6 μl ml−1). The essential oil showed high cytotoxicity on T47D, HEP-G2 and HT-29 cell lines (IC50< 2 μg ml−1). Conclusion: The essential oil of F. carduchorum collected from west of Iran had anti-Candida, larvicidal and cytotoxicity effects and should be further investigated in others in vitro and in vivo experimental models. PMID:26114148

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

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

A simple and rapid radiochemical choline acetyltransferase (ChAT) assay screening test.

PubMed

Shiba, Kazuhiro; Ogawa, Kazuma; Kinuya, Seigo; Yajima, Kazuyoshi; Mori, Hirofumi

2006-10-15

A simple radiochemical choline acetyltransferase (ChAT) assay screening test was developed by measuring for [(3)H]acetylcholine ([(3)H]ACh) formed from 0.2 mM [(3)H]acetyl-coenzyme A ([(3)H]acetyl-CoA) and 1 mM choline by 0.2 mg of rat brain homogenates containing ChAT into 96-well microplates. A simple and rapid procedure for isolating [(3)H]ACh from the incubation mixture into 96-well microplates was achieved by using a sodium tetraphenylboron (Kalibor) solution (in ethyl acetate, 0.75%, w/v) and a hydrophobic liquid scintillator mixture (1:5, v/v, 0.2 mL) as an extraction solvent. The benefits of this radiochemical method using 96-well microplates are as follows: (1) this method is reliable and reproducible; (2) many samples can be examined at the same time by this method; (3) this method is economical and effective in reducing radioactive waste. The development of a new simple radiochemical ChAT assay screening test is the first stage of development of radiolabeled ChAT mapping agent.

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.

Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.

PubMed

Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans Peter; Turecek, Peter L

2017-01-05

BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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.

Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

PubMed Central

2012-01-01

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na+-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline KM with no change in Vmax. As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux

Nonoisotopic assay for the presynaptic choline transporter reveals capacity for allosteric modulation of choline uptake.

PubMed

Ruggiero, Alicia M; Wright, Jane; Ferguson, Shawn M; Lewis, Michelle; Emerson, Katie S; Iwamoto, Hideki; Ivy, Michael T; Holmstrand, Ericka C; Ennis, Elizabeth A; Weaver, C David; Blakely, Randy D

2012-10-17

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux

Effectiveness evaluation of glyphosate oxidation employing the H(2)O(2)/UVC process: toxicity assays with Vibrio fischeri and Rhinella arenarum tadpoles.

PubMed

Junges, Celina M; Vidal, Eduardo E; Attademo, Andrés M; Mariani, Melisa L; Cardell, Leandro; Negro, Antonio C; Cassano, Alberto; Peltzer, Paola M; Lajmanovich, Rafael C; Zalazar, Cristina S

2013-01-01

The H(2)O(2)/UVC process was applied to the photodegradation of a commercial formulation of glyphosate in water. Two organisms (Vibrio fischeri bacteria and Rhinella arenarum tadpoles) were used to investigate the toxicity of glyphosate in samples M(1,) M(2), and M(3) following different photodegradation reaction times (120, 240 and 360 min, respectively) that had differing amounts of residual H(2)O(2). Subsamples of M(1), M(2), and M(3) were then used to create samples M(1,E), M(2,E) and M(3,E) in which the H(2)O(2) had been removed. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in tadpoles to determine possible sub-lethal effects. In V. fischeri, M(1,E), which was collected early in the photodegradation process, caused 52% inhibition, while M(3,E), which was collected at the end of the photodegradation process, caused only 17% inhibition. Survival of tadpoles was 100% in samples M(2), M(3), and in M(1,E), M(2,E) and M(3,E). The lowest percentages of enzymatic inhibition were observed in samples without removal of H(2)O(2): 13.96% (AChE) and 16% (BChE) for M(2), and 24.12% (AChE) and 13.83% (BChE) for M(3). These results show the efficiency of the H(2)O(2)/UVC process in reducing the toxicity of water or wastewater polluted by commercial formulations of glyphosate. According to the ecotoxicity assays, the conditions corresponding to M(2) (11 ± 1 mg a.e. L(-1) glyphosate and 11 ± 1 mg L(-1) H(2)O(2)) could be used as a final point for glyphosate treatment with the H(2)O(2)/UV process.

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.

In vitro antioxidant and anti-cholinesterase activities of Rhizophora mucronata.

PubMed

Suganthy, N; Devi, K Pandima

2016-01-01

Rhizophora mucronata Lam. (Rhizophoraceae), commonly known as Asiatic mangrove, has been used traditionally among Asian countries as folk medicine. This study investigates the cholinesterase inhibitory potential and antioxidant activities of R. mucronata. Rhizophora mucronata leaves were successively extracted using solvents of varying polarity and a dosage of 100-500 µg/ml were used for each assay. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were assessed according to the method of Ellman. In vitro antioxidant activity was assessed using free radical scavenging, reducing power, and metal-chelating activity (duration - 3 months). Total phenolic and flavonoid content were quantified spectrophotometrically. Compound characterization was done using column chromatography, NMR, FTIR, and LC-MS analysis. Methanolic leaf extract (500 µg/ml) exhibited the highest inhibitory activity against AChE (92.73 ± 0.54%) and BuChE (98.98 ± 0.17%), with an IC50 value of 59.31 ± 0.35 and 51.72 ± 0.33 µg/ml, respectively. Among the different solvent extracts, methanolic extract exhibited the highest antioxidant activity with an IC50 value of 47.39 ± 0.43, 401.45 ± 18.52, 80.23 ± 0.70, and 316.47 ± 3.56 µg/ml for DPPH, hydroxyl, nitric oxide radical, and hydrogen peroxide, respectively. Total polyphenolic and flavonoid contents in methanolic extract were observed to be 598.13 ± 1.85 µg of gallic acid equivalent and 48.85 ± 0.70 μg of rutin equivalent/mg of extract. Compound characterization illustrated (+)-catechin as the bioactive compound responsible for cholinesterase inhibitory and antioxidant activities. The presence of rich source of flavonoids, in particular catechin, might be responsible for its cholinesterase inhibitory and antioxidant activities.

[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

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.

The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies

PubMed Central

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

2014-01-01

BACKGROUND AND PURPOSE 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. EXPERIMENTAL APPROACHES 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. KEY RESULTS 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. CONCLUSION AND IMPLICATIONS Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. PMID:24750073

Evaluation of Antioxidant, Antidiabetic and Anticholinesterase Activities of Smallanthus sonchifolius Landraces and Correlation with Their Phytochemical Profiles

PubMed Central

Russo, Daniela; Valentão, Patrícia; Andrade, Paula B.; Fernandez, Eloy C.; Milella, Luigi

2015-01-01

The present study aimed to investigate the phytochemical profile of leaf methanol extracts of fourteen Smallanthus sonchifolius (yacon) landraces and their antioxidant, anticholinesterase and antidiabetic activities that could lead to the finding of more effective agents for the treatment and management of Alzheimer’s disease and diabetes. For this purpose, antioxidant activity was assessed using different tests: ferric reducing ability power (FRAP), 2,2-diphenyl-1-picryl hydrazyl (DPPH), nitric oxide (˙NO) and superoxide (O2˙−) scavenging and lipid peroxidation inhibition assays. Anticholinesterase activity was investigated by quantifying the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, whereas antidiabetic activity was investigated by α-amylase and α-glucosidase inhibition tests. To understand the contribution of metabolites, phytochemical screening was also performed by high performance liquid chromatography-diode array detector (HPLC-DAD) system. Among all, methanol extract of PER09, PER04 and ECU44 landraces exhibited the highest relative antioxidant capacity index (RACI). ECU44 was found to be rich in 4,5-di-O-caffeoylquinic acid (CQA) and 3,5-di-O-CQA and displayed a good α-amylase and α-glucosidase inhibition, showing the lowest IC50 values. Flavonoids, instead, seem to be involved in the AChE and BChE inhibition. The results of this study revealed that the bioactive compound content differences could be determinant for the medicinal properties of this plant especially for antioxidant and antidiabetic activities. PMID:26263984

Evaluation of Antioxidant, Antidiabetic and Anticholinesterase Activities of Smallanthus sonchifolius Landraces and Correlation with Their Phytochemical Profiles.

PubMed

Russo, Daniela; Valentão, Patrícia; Andrade, Paula B; Fernandez, Eloy C; Milella, Luigi

2015-07-31

The present study aimed to investigate the phytochemical profile of leaf methanol extracts of fourteen Smallanthus sonchifolius (yacon) landraces and their antioxidant, anticholinesterase and antidiabetic activities that could lead to the finding of more effective agents for the treatment and management of Alzheimer's disease and diabetes. For this purpose, antioxidant activity was assessed using different tests: ferric reducing ability power (FRAP), 2,2-diphenyl-1-picryl hydrazyl (DPPH), nitric oxide (˙NO) and superoxide (O2˙-) scavenging and lipid peroxidation inhibition assays. Anticholinesterase activity was investigated by quantifying the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, whereas antidiabetic activity was investigated by α-amylase and α-glucosidase inhibition tests. To understand the contribution of metabolites, phytochemical screening was also performed by high performance liquid chromatography-diode array detector (HPLC-DAD) system. Among all, methanol extract of PER09, PER04 and ECU44 landraces exhibited the highest relative antioxidant capacity index (RACI). ECU44 was found to be rich in 4,5-di-O-caffeoylquinic acid (CQA) and 3,5-di-O-CQA and displayed a good α-amylase and α-glucosidase inhibition, showing the lowest IC50 values. Flavonoids, instead, seem to be involved in the AChE and BChE inhibition. The results of this study revealed that the bioactive compound content differences could be determinant for the medicinal properties of this plant especially for antioxidant and antidiabetic activities.

In vitro assay of Staphylococcus aureus enterotoxin A activity in food.

PubMed Central

Rasooly, L; Rose, N R; Shah, D B; Rasooly, A

1997-01-01

Staphylococcus aureus enterotoxin A (SEA) is a leading cause of food poisoning. The current test for functional activity of SEA requires monkeys or kittens. The major drawbacks of animal assays are lack of quantitation, poor reproducibility, low sensitivity, and high cost. In this report we describe and evaluate an alternative assay using T-cell proliferation to measure SEA activity in food. Human and rat lymphocytes proliferate in response to concentrations of SEA as low as 1 pg/ml, well below the pathogenic dose of 100 ng. This proliferation assay is highly sensitive, quantitative, and simple. Nonradioactive assays of T-cell proliferation were also suitable for detecting and measuring SEA, although with a 10-fold lower sensitivity. To evaluate the utility of this assay for food testing, four different food samples were mixed with SEA. In each sample, SEA was detected at a concentration of 1 ng/ml. Heat-inactivated SEA produced no detectable proliferation. These results demonstrate that an in vitro cell proliferation assay is an advantageous alternative to existing animal assays for measuring SEA activity in food. PMID:9172356

α7 nAChR mediated Fas demethylation contributes to prenatal nicotine exposure-induced programmed thymocyte apoptosis in mice.

PubMed

Liu, Han-Xiao; Liu, Sha; Qu, Wen; Yan, Hui-Yi; Wen, Xiao; Chen, Ting; Hou, Li-Fang; Ping, Jie

2017-11-07

This study aimed to investigate the effects of prenatal nicotine exposure (PNE) on thymocyte apoptosis and postnatal immune impairments in vivo and further explore the epigenetic mechanisms of the pro-apoptotic effect of nicotine in vitro . The results showed that PNE caused immune impairments in offspring on postnatal day 49, manifested as increased IL-4 production and an increased IgG1/IgG2a ratio in serum. Enhanced apoptosis of total and CD4+SP thymocytes was observed both in fetus and in offspring. Further, by exposing thymocytes to 0-100 μM of nicotine in vitro for 48 h, we found that nicotine increased α7 nicotinic acetylcholine receptor (nAChR) expression, activated the Fas apoptotic pathway, and promoted thymocyte apoptosis in concentration-dependent manners. In addition, nicotine could induce Tet methylcytosine dioxygenase (TET) 2 expression and Fas promoter demethylation, which can be abolished by TET2 siRNA transfection. Moreover, the α7 nAChR specific antagonist α-bungarotoxin can abrogate nicotine-induced TET2 increase, and the following Fas demethylation and Fas-mediated apoptosis. In conclusion, our findings showed, for the first time, that α7 nAChR activation could induce TET2-mediated Fas demethylation in thymocytes and results in the upregulation of Fas apoptotic pathway, which provide evidence for elucidating the PNE-induced programmed thymocyte apoptosis.

A microplate assay to measure classical and alternative complement activity.

PubMed

Puissant-Lubrano, Bénédicte; Fortenfant, Françoise; Winterton, Peter; Blancher, Antoine

2017-05-01

We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.

A workflow to investigate exposure and pharmacokinetic ...

EPA Pesticide Factsheets

Adverse outcome pathways (AOP) link known population outcomes to a molecular initiating event (MIE) that can be quantified using high-throughput in vitro methods. Practical application of AOPs in chemical-specific risk assessment requires consideration of exposure and absorption, distribution, metabolism, excretion (ADME) properties of chemicals. We developed a conceptual workflow to consider exposure and ADME properties in relationship to an MIE and demonstrated the utility of this workflow using a previously established AOP, acetylcholinesterase (AChE) inhibition. Thirty active chemicals found to inhibit AChE in the ToxCastTM assay were examined with respect to their exposure and absorption potentials, and their ability to cross the blood-brain barrier. Structural similarities of active compounds were compared against structures of inactive compounds to detect possible non-active parents that might have active metabolites. Fifty-two of the 1,029 inactive compounds exhibited a similarity threshold above 75% with their nearest active neighbors. Excluding compounds that may not be absorbed, 22 could be potentially toxic following metabolism. The incorporation of exposure and ADME properties into the conceptual workflow resulted in prioritization of 20 out of 30 active compounds identified in an AChE inhibition assay for further analysis, along with identification of several inactive parent compounds of active metabolites. This qualitative approach can minimize co

Metformin and Its Sulfenamide Prodrugs Inhibit Human Cholinesterase Activity.

PubMed

Markowicz-Piasecka, Magdalena; Sikora, Joanna; Mateusiak, Łukasz; Mikiciuk-Olasik, Elżbieta; Huttunen, Kristiina M

2017-01-01

The results of epidemiological and pathophysiological studies suggest that type 2 diabetes mellitus (T2DM) may predispose to Alzheimer's disease (AD). The two conditions present similar glucose levels, insulin resistance, and biochemical etiologies such as inflammation and oxidative stress. The diabetic state also contributes to increased acetylcholinesterase (AChE) activity, which is one of the factors leading to neurodegeneration in AD. The aim of this study was to assess in vitro the effects of metformin, phenformin, and metformin sulfenamide prodrugs on the activity of human AChE and butyrylcholinesterase (BuChE) and establish the type of inhibition. Metformin inhibited 50% of the AChE activity at micromolar concentrations (2.35  μ mol/mL, mixed type of inhibition) and seemed to be selective towards AChE since it presented low anti-BuChE activity. The tested metformin prodrugs inhibited cholinesterases (ChE) at nanomolar range and thus were more active than metformin or phenformin. The cyclohexyl sulfenamide prodrug demonstrated the highest activity towards both AChE (IC 50  = 890 nmol/mL, noncompetitive inhibition) and BuChE (IC 50  = 28 nmol/mL, mixed type inhibition), while the octyl sulfenamide prodrug did not present anti-AChE activity, but exhibited mixed inhibition towards BuChE (IC 50  = 184 nmol/mL). Therefore, these two bulkier prodrugs were concluded to be the most selective compounds for BuChE over AChE. In conclusion, it was demonstrated that biguanides present a novel class of inhibitors for AChE and BuChE and encourages further studies of these compounds for developing both selective and nonselective inhibitors of ChEs in the future.

Performance characteristics of the ARCHITECT Active-B12 (Holotranscobalamin) assay.

PubMed

Merrigan, Stephen D; Owen, William E; Straseski, Joely A

2015-01-01

Vitamin B12 (cobalamin) is a necessary cofactor in methionine and succinyl-CoA metabolism. Studies estimate the deficiency prevalence as high as 30% in the elderly population. Ten to thirty percent of circulating cobalamin is bound to transcobalamin (holotranscobalamin, holoTC) which can readily enter cells and is therefore considered the bioactive form. The objective of our study was to evaluate the analytical performance of a high-throughput, automated holoTC assay (ARCHITECT i2000(SR) Active-B12 (Holotranscobalamin)) and compare it to other available methods. Manufacturer-specified limits of blank (LoB), detection (LoD), and quantitation (LoQ), imprecision, interference, and linearity were evaluated for the ARCHITECT HoloTC assay. Residual de-identified serum samples were used to compare the ARCHITECT HoloTC assay with the automated AxSYM Active-B12 (Holotranscobalamin) assay (Abbott Diagnostics) and the manual Active-B12 (Holotranscobalamin) Enzyme Immunoassay (EIA) (Axis-Shield Diagnostics, Dundee, Scotland, UK). Manufacturer's claims of LoB, LoD, LoQ, imprecision, interference, and linearity to the highest point tested (113.4 pmol/L) were verified for the ARCHITECT HoloTC assay. Method comparison of the ARCHITECT HoloTC to the AxSYM HoloTC produced the following Deming regression statistics: (ARCHITECT(HoloTc)) = 0.941 (AxSYM(HoloTC)) + 1.2 pmol/L, S(y/x) = 6.4, r = 0.947 (n = 98). Comparison to the Active-B12 EIA produced: (ARCHITECT(HoloTC)) = 1.105 (EIA(Active-B12)) - 6.8 pmol/L, S(y/x) = 11.0, r = 0.950 (n = 221). This assay performed acceptably for LoB, LoD, LoQ, imprecision, interference, linearity and method comparison to the predicate device (AxSYM). An additional comparison to a manual Active-B12 EIA method performed similarly, with minor exceptions. This study determined that the ARCHITECT HoloTC assay is suitable for routine clinical use, which provides a high-throughput alternative for automated testing of this emerging marker of cobalamin

Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) β3 Subunits in Enhanced Functional Expression of Mouse α6β2β3- and α6β4β3-nAChRs*

PubMed Central

Dash, Bhagirathi; Li, Ming D.; Lukas, Ronald J.

2014-01-01

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where “*” indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5–8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′S subunits were substituted for mβ3V9′S subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops “C” (Glu221 and Phe223), “E” (Ser144 and Ser148), and “β2-β3” (Gln94 and Glu101) increased function of mα6mβ2*- (∼2–3-fold) or mα6mβ4* (∼2–4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs. PMID:25028511

Monkey Feeding Assay for Testing Emetic Activity of Staphylococcal Enterotoxin.

PubMed

Seo, Keun Seok

2016-01-01

Staphylococcal enterotoxins (SEs) are unique bacterial toxins that cause gastrointestinal toxicity as well as superantigenic activity. Since systemic administration of SEs induces superantigenic activity leading to toxic shock syndrome that may mimic enterotoxic activity of SEs such as vomiting and diarrhea, oral administration of SEs in the monkey feeding assay is considered as a standard method to evaluate emetic activity of SEs. This chapter summarizes and discusses practical considerations of the monkey feeding assay used in studies characterizing classical and newly identified SEs.

Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

PubMed

Quareshy, Mussa; Uzunova, Veselina; Prusinska, Justyna M; Napier, Richard M

2017-01-01

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.

A protein chip membrane-capture assay for botulinum neurotoxin activity

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

Marconi, Severine; Universite de la Mediterranee-Aix Marseille 2, Faculte de medecine secteur nord, Bd P. Dramard, Marseille F-13916; Ferracci, Geraldine

2008-12-15

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capturemore » providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC{sub 50}s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC{sub 50} of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.« less

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

PubMed Central

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

2016-01-01

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

Toxin activity assays, devices, methods and systems therefor

DOEpatents

Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

2016-04-05

Embodiments of the present invention are directed toward devices, system and method for conducting toxin activity assay using sedimentation. The toxin activity assay may include generating complexes which bind to a plurality of beads in a fluid sample. The complexes may include a target toxin and a labeling agent, or may be generated due to presence of active target toxin and/or labeling agent designed to be incorporated into complexes responsive to the presence of target active toxin. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a lower density than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

Diverse inhibitory actions of quaternary ammonium cholinesterase inhibitors on Torpedo nicotinic ACh receptors transplanted to Xenopus oocytes

PubMed Central

Olivera-Bravo, Silvia; Ivorra, Isabel; Morales, Andrés

2007-01-01

Background and purpose: This work was aimed at comparing and analysing the effects and mechanisms of action of the quaternary ammonium cholinesterase inhibitors (QChEIs) BW284c51, decamethonium and edrophonium, on nicotinic ACh receptor (nAChR) function. Experimental approach: nAChRs purified from Torpedo electroplax were transplanted to oocytes and currents elicited by ACh (IACh) either alone or in presence of these QChEIs were recorded. Key results: None of the QChEIs, by itself, elicited changes in membrane conductance; however, when co-applied with ACh, all of them decreased IACh in a concentration-dependent way. The mechanisms of nAChR inhibition were different for these QChEIs. BW284c51 blockade was non-competitive and voltage-dependent, although it also affected the nH of the dose-response curve. By contrast, decamethonium and edrophonium inhibition, at –60 mV, was apparently competitive and did not modify either desensitisation or nH. Decamethonium effects were voltage-independent and washed out slowly after its removal; by contrast, edrophonium blockade had strong voltage dependence and its effects disappeared quickly after its withdrawal. Analysis of the voltage-dependent blockade indicated that BW284c51 bound to a shallow site into the channel pore, whereas edrophonium bound to a deeper locus. Accordingly, additive inhibitory effects on IACh were found among any pairs of these QChEIs. Conclusions and implications: The tested QChEIs bound to the nAChR at several and different loci, which might account for their complex inhibitory behaviour, acting both as allosteric effectors and, in the case of BW284c51 and edrophonium, as open channel blockers. PMID:17572698

A mutation in the extracellular domain of the α7 nAChR reduces calcium permeability.

PubMed

Colón-Sáez, José O; Yakel, Jerrel L

2014-08-01

The α7 neuronal nicotinic acetylcholine receptor (nAChR) displays the highest calcium permeability among the different subtypes of nAChRs expressed in the mammalian brain and can impact cellular events including neurotransmitter release, second messenger cascades, cell survival, and apoptosis. The selectivity for cations in nAChRs is thought to be achieved in part by anionic residues which are located on either side of the channel mouth and increase relative cationic concentration. Mutagenesis studies have improved our understanding of the role of the second transmembrane domain and the intracellular loop of the channel in ion selectivity. However, little is known about the influence that the extracellular domain (ECD) plays in ion permeation. In the α7 nAChR, it has been found that the ECD contains a ring of ten aspartates (two per subunit) that is believed to face the lumen of the pore and could attract cations for permeation. Using mutagenesis and a combination of electrophysiology and imaging techniques, we tested the possible involvement of these aspartate residues in the calcium permeability of the rat α7 nAChR. We found that one of these residues (the aspartate at position 44) appears to be essential since mutating it to alanine resulted in a decrease in amplitude for both whole cell and single-channel responses and in the complete disappearance of detectable calcium changes in most cells, which indicates that the ECD of the α7 nAChR plays a key role in calcium permeation.

A mutation in the extracellular domain of the α7 nAChR reduces calcium permeability

PubMed Central

Colón-Sáez, José O.

2013-01-01

The α7 neuronal nicotinic acetylcholine receptor (nAChR) displays the highest calcium permeability among the different subtypes of nAChRs expressed in the mammalian brain and can impact cellular events including neurotransmitter release, second messenger cascades, cell survival, and apoptosis. The selectivity for cations in nAChRs is thought to be achieved in part by anionic residues which are located on either side of the channel mouth and increase relative cationic concentration. Mutagenesis studies have improved our understanding of the role of the second transmembrane domain and the intracellular loop of the channel in ion selectivity. However, little is known about the influence that the extracellular domain (ECD) plays in ion permeation. In the α7 nAChR, it has been found that the ECD contains a ring of ten aspartates (two per subunit) that is believed to face the lumen of the pore and could attract cations for permeation. Using mutagenesis and a combination of electrophysiology and imaging techniques, we tested the possible involvement of these aspartate residues in the calcium permeability of the rat α7 nAChR. We found that one of these residues (the aspartate at position 44) appears to be essential since mutating it to alanine resulted in a decrease in amplitude for both whole cell and single-channel responses and in the complete disappearance of detectable calcium changes in most cells, which indicates that the ECD of the α7 nAChR plays a key role in calcium permeation. PMID:24177919

Microplate-based filter paper assay to measure total cellulase activity.

PubMed

Xiao, Zhizhuang; Storms, Reginald; Tsang, Adrian

2004-12-30

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%. 2004 Wiley Periodicals, Inc.

Comparative study on collagen-binding enzyme-linked immunosorbent assay and ristocetin cofactor activity assays for detection of functional activity of von Willebrand factor.

PubMed

Turecek, Peter L; Siekmann, Jürgen; Schwarz, Hans Peter

2002-04-01

For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP).

PubMed

Bainbridge, Travis W; Dunshee, Diana Ronai; Kljavin, Noelyn M; Skelton, Nicholas J; Sonoda, Junichiro; Ernst, James A

2017-10-02

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

The acetylcholinesterase inhibitor BW284c51 is a potent blocker of Torpedo nicotinic AchRs incorporated into the Xenopus oocyte membrane

PubMed Central

Olivera-Bravo, Silvia; Ivorra, Isabel; Morales, Andrés

2004-01-01

This work was aimed to determine if 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51), the most selective acetylcholinesterase inhibitor (AchEI), affects the nicotinic acetylcholine (Ach) receptor (AchR) function. Purified Torpedo nicotinic AchRs were injected into Xenopus laevis oocytes and BW284c51 effects on Ach- and carbamylcholine (Cch)-elicited currents were assessed using the voltage-clamp technique. BW284c51 (up to 1 mM) did not evoke any change in the oocyte membrane conductance. When BW284c51 (10 pM–100 μM) and Ach were coapplied, Ach-evoked currents (IAch) were reversibly inhibited in a concentration-dependent manner (Hill coefficient, 1; IC50, 0.2–0.5 μM for 0.1–1000 μM Ach). Cch-elicited currents showed a similar inhibition by BW284c51. IAch blockade by BW284c51 showed a strong voltage dependence, being only apparent at hyperpolarising potentials. BW284c51 also enhanced IAch desensitisation. BW284c51 changed the Ach concentration-dependence curve of Torpedo AchR response from two-site to single-site kinetics, without noticeably affecting the EC50 value. The BW284c51 blocking effect was highly selective for nicotinic over muscarinic receptors. BW284c51 inhibition potency was stronger than that of tacrine, and similar to that of d-tubocurarine (d-TC). Coapplication of BW284c51 with either tacrine or d-TC revealed synergistic inhibitory effects. Our results indicate that BW284c51 antagonises nicotinic AchRs in a noncompetitive way by blocking the receptor channel, and possibly by other, yet unknown, mechanisms. Therefore, besides acting as a selective AchEI, BW284c51 constitutes a powerful and reversible blocker of nicotinic AchRs that might be used as a valuable tool for understanding their function. PMID:15644872

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.

Enzyme activity assays within microstructured optical fibers enabled by automated alignment.

PubMed

Warren-Smith, Stephen C; Nie, Guiying; Schartner, Erik P; Salamonsen, Lois A; Monro, Tanya M

2012-12-01

A fluorescence-based enzyme activity assay has been demonstrated within a small-core microstructured optical fiber (MOF) for the first time. To achieve this, a reflection-based automated alignment system has been developed, which uses feedback and piezoelectric actuators to maintain optical alignment. The auto-alignment system provides optical stability for the time required to perform an activity assay. The chosen assay is based on the enzyme proprotein convertase 5/6 (PC6) and has important applications in women's health.

A homogeneous cell-based assay for measurement of endogenous paraoxonase 1 activity.

PubMed

Ahmad, Syed; Carter, Jade J; Scott, John E

2010-05-01

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. To identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in medium and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells, and tolerance to dimethyl sulfoxide and serum. Aspirin, quercetin, and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and Western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel homogeneous assay for detection of endogenous PON1 expression has been developed and is amenable to high-throughput screening for the identification of small molecules that enhance PON1 expression. 2010 Elsevier Inc. All rights reserved.

The α7-nACh nicotinic receptor and its role in memory and selected diseases of the central nervous system.

PubMed

Baranowska, Urszula; Wiśniewska, Róża Julia

2017-07-30

α7-nACh is one of the major nicotinic cholinergic receptor subtypes found in the brain. It is broadly expressed in the hippocampal and cortical neurons, the regions which play a key role in memory formation. Although α7-nACh receptors may serve as postsynaptic receptors mediating classical neurotransmission, they usually function as presynaptic modulators responsible for the release of other neurotransmitters, such as glutamate, γ-aminobutyric acid, dopamine, and norepinephrine. They can, therefore, affect a wide array of neurobiological functions. In recent years, research has found that a large number of agonists and positive allosteric modulators of α7-nAChR induce beneficial effects on learning and memory. Consistently, mice deficient in chrna7 (the gene encoding α7-nAChR protein), are characterized by memory deficits. In addition, decreased expression and function of α7-nAChR is associated agoniwith many neurological diseases including schizophrenia, bipolar disorder, learning disability, attention deficit hyperactivity disorder, Alzheimer disease, autism, and epilepsy. In the recent years many animal experiments and clinical trials using α7-nAChR ligands were conducted. The results of these studies strongly indicate that agonists and positive allosteric modulators of α7-nAChR are promising therapeutic agents for diseases associated with cognitive deficits.

Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

PubMed

Chanput, Wasaporn; Krueyos, Narumol; Ritthiruangdej, Pitiporn

2016-11-01

The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular recognition of thiaclopride by Aplysia californica AChBP: new insights from a computational investigation

NASA Astrophysics Data System (ADS)

Alamiddine, Zakaria; Selvam, Balaji; Cerón-Carrasco, José P.; Mathé-Allainmat, Monique; Lebreton, Jacques; Thany, Steeve H.; Laurent, Adèle D.; Graton, Jérôme; Le Questel, Jean-Yves

2015-12-01

The binding of thiaclopride (THI), a neonicotinoid insecticide, with Aplysia californica acetylcholine binding protein ( Ac-AChBP), the surrogate of the extracellular domain of insects nicotinic acetylcholine receptors, has been studied with a QM/QM' hybrid methodology using the ONIOM approach (M06-2X/6-311G(d):PM6). The contributions of Ac-AChBP key residues for THI binding are accurately quantified from a structural and energetic point of view. The importance of water mediated hydrogen-bond (H-bond) interactions involving two water molecules and Tyr55 and Ser189 residues in the vicinity of the THI nitrile group, is specially highlighted. A larger stabilization energy is obtained with the THI- Ac-AChBP complex compared to imidacloprid (IMI), the forerunner of neonicotinoid insecticides. Pairwise interaction energy calculations rationalize this result with, in particular, a significantly more important contribution of the pivotal aromatic residues Trp147 and Tyr188 with THI through CH···π/CH···O and π-π stacking interactions, respectively. These trends are confirmed through a complementary non-covalent interaction (NCI) analysis of selected THI- Ac-AChBP amino acid pairs.

Measurement of filter paper activities of cellulase with microplate-based assay.

PubMed

Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

2016-01-01

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC.

Measurement of filter paper activities of cellulase with microplate-based assay

PubMed Central

Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

2015-01-01

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC. PMID:26858572

Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore

PubMed Central

Talley, Todd T.; Harel, Michal; Hibbs, Ryan E.; Radić, Zoran; Tomizawa, Motohiro; Casida, John E.; Taylor, Palmer

2008-01-01

Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 Å in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids. PMID:18477694

Monoterpenoids (thymol, carvacrol and S-(+)-linalool) with anesthetic activity in silver catfish (Rhamdia quelen): evaluation of acetylcholinesterase and GABAergic activity

PubMed Central

Bianchini, A.E.; Garlet, Q.I.; da Cunha, J.A.; Bandeira, G.; Brusque, I.C.M.; Salbego, J.; Heinzmann, B.M.; Baldisserotto, B.

2017-01-01

This study evaluated the anesthetic potential of thymol and carvacrol, and their influence on acetylcholinesterase (AChE) activity in the muscle and brain of silver catfish (Rhamdia quelen). The AChE activity of S-(+)-linalool was also evaluated. We subsequently assessed the effects of thymol and S-(+)-linalool on the GABAergic system. Fish were exposed to thymol and carvacrol (25, 50, 75, and 100 mg/L) to evaluate time for anesthesia and recovery. Both compounds induced sedation at 25 mg/L and anesthesia with 50–100 mg/L. However, fish exposed to carvacrol presented strong muscle contractions and mortality. AChE activity was increased in the brain of fish at 50 mg/L carvacrol and 100 mg/L thymol, and decreased in the muscle at 100 mg/L carvacrol. S-(+)-linalool did not alter AChE activity. Anesthesia with thymol was reversed by exposure to picrotoxin (GABAA antagonist), similar to the positive control propofol, but was not reversed by flumazenil (antagonist of benzodiazepine binding site), as observed for the positive control diazepam. Picrotoxin did not reverse the effect of S-(+)-linalool. Thymol exposure at 50 mg/L is more suitable than carvacrol for anesthesia in silver catfish, because this concentration did not cause any mortality or interference with AChE activity. Thymol interacted with GABAA receptors, but not with the GABAA/benzodiazepine site. In contrast, S-(+)-linalool did not act in GABAA receptors in silver catfish. PMID:29069225

Monoterpenoids (thymol, carvacrol and S-(+)-linalool) with anesthetic activity in silver catfish (Rhamdia quelen): evaluation of acetylcholinesterase and GABAergic activity.

PubMed

Bianchini, A E; Garlet, Q I; da Cunha, J A; Bandeira, G; Brusque, I C M; Salbego, J; Heinzmann, B M; Baldisserotto, B

2017-10-19

This study evaluated the anesthetic potential of thymol and carvacrol, and their influence on acetylcholinesterase (AChE) activity in the muscle and brain of silver catfish (Rhamdia quelen). The AChE activity of S-(+)-linalool was also evaluated. We subsequently assessed the effects of thymol and S-(+)-linalool on the GABAergic system. Fish were exposed to thymol and carvacrol (25, 50, 75, and 100 mg/L) to evaluate time for anesthesia and recovery. Both compounds induced sedation at 25 mg/L and anesthesia with 50-100 mg/L. However, fish exposed to carvacrol presented strong muscle contractions and mortality. AChE activity was increased in the brain of fish at 50 mg/L carvacrol and 100 mg/L thymol, and decreased in the muscle at 100 mg/L carvacrol. S-(+)-linalool did not alter AChE activity. Anesthesia with thymol was reversed by exposure to picrotoxin (GABAA antagonist), similar to the positive control propofol, but was not reversed by flumazenil (antagonist of benzodiazepine binding site), as observed for the positive control diazepam. Picrotoxin did not reverse the effect of S-(+)-linalool. Thymol exposure at 50 mg/L is more suitable than carvacrol for anesthesia in silver catfish, because this concentration did not cause any mortality or interference with AChE activity. Thymol interacted with GABAA receptors, but not with the GABAA/benzodiazepine site. In contrast, S-(+)-linalool did not act in GABAA receptors in silver catfish.

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.

Rat nicotinic ACh receptor α7 and β2 subunits co-assemble to form functional heteromeric nicotinic receptor channels

PubMed Central

Khiroug, Serguei S; Harkness, Patricia C; Lamb, Patricia W; Sudweeks, Sterling N; Khiroug, Leonard; Millar, Neil S; Yakel, Jerrel L

2002-01-01

Rat hippocampal interneurons express diverse subtypes of functional nicotinic acetylcholine receptors (nAChRs), including α7-containing receptors that have properties unlike those expected for homomeric α7 nAChRs. We previously reported a strong correlation between expression of the α7 and of the β2 subunits in individual neurons. To explore whether co-assembly of the α7 and β2 subunits might occur, these subunits were co-expressed in Xenopus oocytes and the functional properties of heterologously expressed nAChRs were characterized by two-electrode voltage clamp. Co-expression of the β2 subunit, both wild-type and mutant forms, with the α7 subunit significantly slowed the rate of nAChR desensitization and altered the pharmacological properties. Whereas ACh, carbachol and choline were full or near-full agonists for homomeric α7 receptor channels, both carbachol and choline were only partial agonists in oocytes expressing both α7 and β2 subunits. In addition the EC50 values for all three agonists significantly increased when the β2 subunit was co-expressed with the α7 subunit. Co-expression with the β2 subunit did not result in any significant change in the current-voltage curve. Biochemical evidence for the co-assembly of the α7 and β2 subunits was obtained by co-immunoprecipitation of these subunits from transiently transfected human embryonic kidney (TSA201) cells. These data provide direct biophysical and molecular evidence that the nAChR α7 and β2 subunits co-assemble to form a functional heteromeric nAChR with functional and pharmacological properties different from those of homomeric α7 channels. This co-assembly may help to explain nAChR channel diversity in rat hippocampal interneurons, and perhaps in other areas of the nervous system. PMID:11956333

Antioxidant and anticholinesterase activities of five wild mushroom species with total bioactive contents.

PubMed

Tel, Gulsen; Ozturk, Mehmet; Duru, Mehmet E; Turkoglu, Aziz

2015-06-01

Recently, mushrooms are interesting natural products to be investigated due to exhibiting various bioactivities. This study determines the antioxidant and anticholinesterase activities of various extracts of five wild mushroom species. In addition, the total bioactive contents, namely, ascorbic acid, β-carotene, and lycopene along with phenolic and flavonoid contents were also determined spectrophotometrically. Antioxidant activity was tested by using five complementary tests; namely, β-carotene-linoleic acid, DPPH(•) scavenging, ABTS(•+) scavenging, cupric-reducing antioxidant capacity (CUPRAC), and metal chelating assays. The in vitro anticholinesterase activity was tested against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the Ellman method. The spectrophotometric methods were used to determine the total phenolic, flavonoid, ascorbic acid, β-carotene, and lycopene contents. The current study has shown that ethyl acetate extracts of Ganoderma lucidum (Curtis) P. Karst (IC50: 1.55 ± 0.05 µg/mL) and Funalia trogii (Berk.) Bondartsev & Singer (IC50: 4.31 ± 0.18 µg/mL) exhibited good lipid peroxidation inhibitory activity. The DPPH, ABTS, and CUPRAC assays supported this activity. The ethyl acetate and methanol extracts of Funalia trogii and Ganoderma lucidum indicated good anticholinesterase activity. Ganoderma lucidum had rich phenolic and flavonoid contents, indicating 98.67 ± 0.32 mg PEs/g extract and 160.38 ± 1.25 mg QEs/g extract, respectively. The results demonstrate that some of the mushroom species tested herein could be used in food and pharmaceutical industries as natural antioxidants.

Memantine Inhibits α3β2-nAChRs-Mediated Nitrergic Neurogenic Vasodilation in Porcine Basilar Arteries

PubMed Central

Wu, Celeste Yin-Chieh; Chen, Po-Yi; Chen, Mei-Fang; Kuo, Jon-Son; Lee, Tony Jer-Fu

2012-01-01

Memantine, an NMDA receptor antagonist used for treatment of Alzheimer’s disease (AD), is known to block the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS). In the present study, we examined by wire myography if memantine inhibited α3β2-nAChRs located on cerebral perivascular sympathetic nerve terminals originating in the superior cervical ganglion (SCG), thus, leading to inhibition of nicotine-induced nitrergic neurogenic dilation of isolated porcine basilar arteries. Memantine concentration-dependently blocked nicotine-induced neurogenic dilation of endothelium-denuded basilar arteries without affecting that induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, memantine significantly inhibited nicotine-elicited inward currents in Xenopous oocytes expressing α3β2-, α7- or α4β2-nAChR, and nicotine-induced calcium influx in cultured rat SCG neurons. These results suggest that memantine is a non-specific antagonist for nAChR. By directly inhibiting α3β2-nAChRs located on the sympathetic nerve terminals, memantine blocks nicotine-induced neurogenic vasodilation of the porcine basilar arteries. This effect of memantine is expected to reduce the blood supply to the brain stem and possibly other brain regions, thus, decreasing its clinical efficacy in the treatment of Alzheimer’s disease. PMID:22792283

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

PubMed Central

Tilley, Derek; Levit, Irina; Samis, John A.

2012-01-01

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre

Measurement of factor v activity in human plasma using a microplate coagulation assay.

PubMed

Tilley, Derek; Levit, Irina; Samis, John A

2012-09-09

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

Mechanisms of flow and ACh-induced dilation in rat soleus arterioles are altered by hindlimb unweighting

NASA Technical Reports Server (NTRS)

Schrage, William G.; Woodman, Christopher R.; Laughlin, M. Harold

2002-01-01

The purpose of this study was to test the hypothesis that endothelium-dependent dilation (flow-induced dilation and ACh-induced dilation) in rat soleus muscle arterioles is impaired by hindlimb unweighting (HLU). Male Sprague-Dawley rats (approximately 300 g) were exposed to HLU or weight-bearing control (Con) conditions for 14 days. Soleus first-order (1A) and second-order (2A) arterioles were isolated, cannulated, and exposed to step increases in luminal flow at constant pressure. Flow-induced dilation was not impaired by HLU in 1A or 2A arterioles. The cyclooxygenase inhibitor indomethacin (Indo; 50 microM) did not alter flow-induced dilation in 1As or 2As. Inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-L-arginine (L-NNA; 300 microM) reduced flow-induced dilation by 65-70% in Con and HLU 1As. In contrast, L-NNA abolished flow-induced dilation in 2As from Con rats but had no effect in HLU 2As. Combined treatment with L-NNA + Indo reduced tone in 1As and 2As from Con rats, but flow-induced dilation in the presence of L-NNA + Indo was not different from responses without inhibitors in either Con or HLU 1As or 2As. HLU also did not impair ACh-induced dilation (10(-9)-10(-4) M) in soleus 2As. L-NNA reduced ACh-induced dilation by approximately 40% in Con 2As but abolished dilation in HLU 2As. Indo did not alter ACh-induced dilation in Con or HLU 2As, whereas combined treatment with L-NNA + Indo abolished ACh-induced dilation in 2As from both groups. We conclude that flow-induced dilation (1As and 2As) was preserved after 2 wk HLU, but HLU decreased the contribution of NOS in mediating flow-induced dilation and increased the contribution of a NOS- and cyclooxygenase-independent mechanism (possibly endothelium-derived hyperpolarizing factor). In soleus 2As, ACh-induced dilation was preserved after 2-wk HLU but the contribution of NOS in mediating ACh-induced dilation was increased.

Oximes: Inhibitors of Human Recombinant Acetylcholinesterase. A Structure-Activity Relationship (SAR) Study

PubMed Central

Sepsova, Vendula; Karasova, Jana Zdarova; Korabecny, Jan; Dolezal, Rafael; Zemek, Filip; Bennion, Brian J.; Kuca, Kamil

2013-01-01

Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring. PMID:23959117

Spacetime Non-Commutativity Corrections to the Cardy-Verlinde Formula of Achúcarro-Ortiz Black Hole

NASA Astrophysics Data System (ADS)

Setare, M. R.

2007-02-01

In this letter we compute the corrections to the Cardy-Verlinde formula of Achúcarro-Ortiz black hole, which is the most general two-dimensional black hole derived from the three-dimensional rotating Banados-Teitelboim-Zanelli black hole. These corrections stem from the space non-commutativity. We show that in non-commutative case, non-rotating Achúcarro-Ortiz black hole in contrast with commutative case has two horizons.

In vitro antioxidant and cholinesterase inhibitory activities of Elatostema papillosum leaves and correlation with their phytochemical profiles: a study relevant to the treatment of Alzheimer's disease.

PubMed

Ali Reza, A S M; Hossain, Mohammad Shahadat; Akhter, Sharmin; Rahman, Md Rezanur; Nasrin, Mst Samima; Uddin, Md Josim; Sadik, Golam; Khurshid Alam, A H M

2018-04-05

Alzheimer's disease (AD), one of the major causes of dementia, is an overwhelming neurodegenerative disease that particularly affects the brain, leading to memory loss and impairment of language and judgment capacity. The aim of the present study was to investigate the antioxidant and anticholinesterase properties of the leaves of Elatostema papillosum (EPL) and correlate with their phytochemical profiles, which are relevant to the treatment of AD. The dried coarse powder of EPL was extracted with 80% methanol (EPL-M80) by cold extraction method. The resultant EPL-M80 was assessed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity by the Ellman method. The antioxidant activity was determined by DPPH (1, 1-diphenyl-2-picrylhydrazyl) and hydroxyl radical scavenging assays. Quantitative phytochemical (phenolic and flavonoid contents) analysis of endogenous substances in EPL-M80 was performed by standard spectrophotometric methods. EPL-M80 significantly (p < 0.05) inhibited AChE and BChE activity with IC 50 of 165.40 ± 4.01 and 213.81 ± 3.57 μg/mL, respectively in a dose-dependent manner. Additionally, EPL-M80 exhibited strong radical scavenging activity against DPPH (IC 50 = 32.35 ± 0.68 μg/mL) and hydroxyl radical (IC 50 = 19.67 ± 1.42 μg/mL) when compared to that of standards. EPL-M80 was found to be rich in phenolic (23.74 mg gallic acid equivalent/g of dry extract) and flavonoid (31.18 mg quercetin equivalent/g of dry extract) content. Furthermore, a positive correlation (p < 0.001) was observed between the total phenolics and antioxidant as well as the anticholinesterase potential. The marked inhibition of AChE and BChE, and potent antioxidant activity of the leaves of Elatostema papillosum highlight its potential to provide an effective treatment for AD.

Assessment of erythrocyte acetylcholine esterase activities in painters.

PubMed

Khan, Mohd Imran; Mahdi, Abbas Ali; Islam, Najmul; Rastogi, Subodh Kumar; Negi, M P S

2009-04-01

Thirty-five male painters in the age group of 20-50 years occupationally engaged in domestic and commercial painting for 5-12 years having blood lead levels (BLL) AChE) levels both in plasma and red blood cell (RBC) lysate. BLL were determined using a graphite furnace atomic absorption spectrometer. The results showed that BLL were 7.7 times higher in the painters as compared with that of the control group. Significant decreases in RBC and plasma AChE were observed in the exposed group in comparison with controls. RBC and plasma AChE showed a decrease of 18.4% and 18%, respectively, in the exposed group. The findings also indicated a significant negative correlation of both RBC and plasma AChE activities with BLL. The marked reduction observed in both RBC and plasma AChE activity may account for disruption of cholinergic function and result in neurotoxicity among the painters.

Compounds from Sedum caeruleum with antioxidant, anticholinesterase, and antibacterial activities.

PubMed

Bensouici, Chawki; Kabouche, Ahmed; Karioti, Anastasia; Öztürk, Mehmet; Duru, Mehmet Emin; Bilia, Anna Rita; Kabouche, Zahia

2016-01-01

This is the first study on the phytochemistry, antioxidant, anticholinesterase, and antibacterial activities of Sedum caeruleum L. (Crassulaceae). The objective of this study is to isolate the secondary metabolites and determine the antioxidant, anticholinesterase, and antibacterial activities of S. caeruleum. Six compounds (1-6) were isolated from the extracts of S. caeruleum and elucidated using UV, 1D-, 2D-NMR, and MS techniques. Antioxidant activity was investigated using DPPH(•), CUPRAC, and ferrous-ions chelating assays. Anticholinesterase activity was determined against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the Ellman method. Antibacterial activity was performed according to disc diffusion and minimum inhibitory concentration (MIC) methods. Isolated compounds were elucidated as ursolic acid (1), daucosterol (2), β-sitosterol-3-O-β-D-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6). The butanol extract exhibited highest antioxidant activity in all tests (IC50 value: 28.35 ± 1.22 µg/mL in DPPH assay, IC50 value: 40.83 ± 2.24 µg/L in metal chelating activity, and IC50 value: 23.52 ± 0.44 µg/L in CUPRAC), and the highest BChE inhibitory activity (IC50 value: 36.89 ± 0.15 µg/L). Moreover, the chloroform extract mildly inhibited (MIC value: 80 µg/mL) the growth of all the tested bacterial strains. Ursolic acid (1), daucosterol (2), β-sitosterol-3-O-β-D-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6) were isolated from Sedum caeruleum for the first time. In addition, a correlation was observed between antioxidant and anticholinesterase activities of bioactive ingredients of this plant.

Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

PubMed Central

Mukhtasimova, Nuriya; daCosta, Corrie J.B.

2016-01-01

The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist–receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate

Nicotine/cigarette smoke promotes metastasis of pancreatic cancer through α7nAChR-mediated MUC4 upregulation.

PubMed

Momi, N; Ponnusamy, M P; Kaur, S; Rachagani, S; Kunigal, S S; Chellappan, S; Ouellette, M M; Batra, S K

2013-03-14

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared with the normal pancreas, and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette smoke extract and nicotine, which is the major component of CS, significantly upregulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via the α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. In addition, we demonstrated that nicotine-mediated MUC4 upregulation promotes the PC cell migration through the activation of the downstream effectors, such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in vivo studies showed a marked increase in the mean pancreatic tumor weight (low dose (100 mg/m(3) total suspended particulate (TSP)), P=0.014; high dose (247 mg/m(3) TSP), P=0.02) and significant tumor metastasis to various distant organs in the CS-exposed mice, orthotopically implanted with luciferase-transfected PC cells, as compared with the sham controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine (low dose, 155.88±35.96 ng/ml; high dose, 216.25±29.95 ng/ml) and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings

Understanding the molecular mechanism of aryl acylamidase activity of acetylcholinesterase - An in silico study.

PubMed

Chinnadurai, Raj Kumar; Saravanaraman, Ponne; Boopathy, Rathanam

2015-08-15

Acetylcholinesterase (AChE) exhibits two different activities, namely esterase and aryl acylamidase (AAA). Unlike esterase, AAA activity of AChE is inhibited by the active site inhibitors while remaining unaffected by the peripheral anionic site inhibitors. This differential inhibitory pattern of active and peripheral anionic site inhibitors on the AAA activity remains unanswered. To answer this, we investigated the mechanism of binding and trafficking of AAA substrates using in silico tools. Molecular docking of serotonin and AAA substrates (o-nitroacetanilide, and o-nitrotrifluoroacetanilide,) onto AChE shows that these compounds bind at the side door of AChE. Thus, we conceived that the AAA substrates prefer the side door to reach the active site for their catalysis. Further, steered molecular dynamics simulations show that the force required for binding and trafficking of the AAA substrate through the side door is comparatively lesser than their dissociation (900kJ/mol/nm). Among the two substrates, o-nitrotrifluoroacetanilide required lesser force (380kJ/mol/nm) than o-nitroacetanilide the (550kJ/mol/nm) for its binding, thus validating o-nitrotrifluoroacetanilide as a better substrate. With these observations, we resolve that the AAA activity of AChE is mediated through its side door. Therefore, binding of PAS inhibitors at the main door of AChE remain ineffective against AAA activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Green-synthesized CdS nano-pesticides: Toxicity on young instars of malaria vectors and impact on enzymatic activities of the non-target mud crab Scylla serrata.

PubMed

Sujitha, Vasu; Murugan, Kadarkarai; Dinesh, Devakumar; Pandiyan, Amuthvalli; Aruliah, Rajasekar; Hwang, Jiang-Shiou; Kalimuthu, Kandasamy; Panneerselvam, Chellasamy; Higuchi, Akon; Aziz, Al Thabiani; Kumar, Suresh; Alarfaj, Abdullah A; Vaseeharan, Baskaralingam; Canale, Angelo; Benelli, Giovanni

2017-07-01

Currently, nano-formulated mosquito larvicides have been widely proposed to control young instars of malaria vector populations. However, the fate of nanoparticles in the aquatic environment is scarcely known, with special reference to the impact of nanoparticles on enzymatic activity of non-target aquatic invertebrates. In this study, we synthesized CdS nanoparticles using a green protocol relying on the cheap extract of Valoniopsis pachynema algae. CdS nanoparticles showed high toxicity on young instars of the malaria vectors Anopheles stephensi and A. sundaicus. The antimalarial activity of the nano-synthesized product against chloroquine-resistant (CQ-r) Plasmodium falciparum parasites was investigated. From a non-target perspective, we focused on the impact of this novel nano-pesticide on antioxidant enzymes acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities of the mud crab Scylla serrata. The characterization of nanomaterials was carried out by UV-vis and FTIR spectroscopy, as well as SEM and XRD analyses. In mosquitocidal assays, LC 50 of V. pachynema-synthesized CdS nanoparticles on A. stephensi ranged from 16.856 (larva I), to 30.301μg/ml (pupa), while for An. sundaicus they ranged from 13.584 to 22.496μg/ml. The antiplasmodial activity of V. pachynema extract and CdS nanoparticles was evaluated against CQ-r and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC 50 of V. pachynema extract was 58.1μg/ml (CQ-s) and 71.46μg/ml (CQ-r), while nano-CdS IC 50 was 76.14μg/ml (CQ-s) and 89.21μg/ml (CQ-r). In enzymatic assays, S. serrata crabs were exposed to sub-lethal concentrations, i.e. 4, 6 and 8μg/ml of CdS nanoparticles, assessing changes in GST and AChE activity after 16days. We observed significantly higher activity of GST, if compared to the control, during the whole experiment period. In addition, a single treatment with CdS nanoparticles led to a significant decrease in AChE activity over time. The toxicity of Cd

Discovering New Acetylcholinesterase Inhibitors by Mining the Buzhongyiqi Decoction Recipe Data.

PubMed

Cui, Lu; Wang, Yu; Liu, Zhihong; Chen, Hongzhuan; Wang, Hao; Zhou, Xinxin; Xu, Jun

2015-11-23

Myasthenia gravis (MG) is a neuromuscular disease that is conventionally treated with acetylcholinesterase (AChE) inhibitors, which may not fully remove the symptom for many reasons. When AChE inhibitors do not work, Chinese patients turn to Chinese medicine, such as the Buzhongyiqi decoction (BD), to treat MG. By elucidating the relations between the herbs of the Buzhongyiqi decoction recipe and AChE inhibitors with structure-based and ligand-based drug design methods and chemoinformatics approaches, we have found the key active components of BD. Using these key active components as templates, we have discovered five new AChE inhibitors through virtual screening of a commercial compound library. The new AChE inhibitors have been confirmed with Ellman assays. This study demonstrates that lead identification can be inspired by elucidating Chinese medicine. Since BD is a mixture, further studies against other drug targets are needed.

Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

PubMed

Lillo, Mauricio A; Gaete, Pablo S; Puebla, Mariela; Ardiles, Nicolás M; Poblete, Inés; Becerra, Alvaro; Simon, Felipe; Figueroa, Xavier F

2018-04-01

Na + -Ca 2+ exchanger (NCX) contributes to control the intracellular free Ca 2+ concentration ([Ca 2+ ] i ), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca 2+ uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively. In addition, both blockers abolished the NO production activated by ACh in intact mesenteric arteries. In contrast, the inhibition of NCXrm did not affect the vasodilator responses induced by the Ca 2+ ionophore, ionomycin, and the NO donor, S-nitroso- N-acetylpenicillamine. Furthermore, SEA0400, KB-R7943, and a small interference RNA directed against NCX1 blunted the increase in [Ca 2+ ] i induced by ACh or ATP in cultured endothelial cells. The analysis by proximity ligation assay showed that the NO-synthesizing enzyme, eNOS, and NCX1 were associated in endothelial cell caveolae of intact mesenteric resistance arteries. These results indicate that the activation of NCXrm has a central role in Ca 2+ -mediated vasodilation initiated by ACh in endothelial cells of resistance arteries.-Lillo, M. A., Gaete, P. S., Puebla, M., Ardiles, N. M., Poblete, I., Becerra, A., Simon, F., Figueroa, X. F. Critical contribution of Na + -Ca 2+ exchanger to the Ca 2+ -mediated vasodilation activated in endothelial cells of resistance arteries.

Acetylcholinesterase Activity and Neurodevelopment in Boys and Girls

PubMed Central

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

2013-01-01

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

Going up in Smoke? A Review of nAChRs-based Treatment Strategies for Improving Cognition in Schizophrenia

PubMed Central

Boggs, Douglas L.; Carlson, Jon; Cortes-Briones, Jose; Krystal, John H.; D’Souza, D. Cyril

2015-01-01

Cognitive impairment is known to be a core deficit in schizophrenia. Existing treatments for schizophrenia have limited efficacy against cognitive impairment. The ubiquitous use of nicotine in this population is thought to reflect an attempt by patients to self-medicate certain symptoms associated with the illness. Concurrently there is evidence that nicotinic receptors that have lower affinity for nicotine are more important in cognition. Therefore, a number of medications that target nicotinic acetylcholine receptors (nAChRs) have been tested or are in development. In this article we summarize the clinical evidence of nAChRs dysfunction in schizophrenia and review clinical studies testing either nicotine or nicotinic medications for the treatment of cognitive impairment in schizophrenia. Some evidence suggests beneficial effects of nAChRs based treatments for the attentional deficits associated with schizophrenia. Standardized cognitive test batteries have failed to capture consistent improvements from drugs acting at nAChRs. However, more proximal measures of brain function, such as ERPs relevant to information processing impairments in schizophrenia, have shown some benefit. Further work is necessary to conclude that nAChRs based treatments are of clinical utility in the treatment of cognitive deficits of schizophrenia. PMID:24345265

Acetylcholinesterase-independent protective effects of huperzine A against iron overload-induced oxidative damage and aberrant iron metabolism signaling in rat cortical neurons.

PubMed

Tao, Ling-Xue; Huang, Xiao-Tian; Chen, Yu-Ting; Tang, Xi-Can; Zhang, Hai-Yan

2016-11-01

Iron dyshomeostasis is one of the primary causes of neuronal death in Alzheimer's disease (AD). Huperzine A (HupA), a natural inhibitor of acetylcholinesterase (AChE), is a licensed anti-AD drug in China and a nutraceutical in the United Sates. Here, we investigated the protective effects of HupA against iron overload-induced injury in neurons. Rat cortical neurons were treated with ferric ammonium citrate (FAC), and cell viability was assessed with MTT assays. Reactive oxygen species (ROS) assays and adenosine triphosphate (ATP) assays were performed to assess mitochondrial function. The labile iron pool (LIP) level, cytosolic-aconitase (c-aconitase) activity and iron uptake protein expression were measured to determine iron metabolism changes. The modified Ellman's method was used to evaluate AChE activity. HupA significantly attenuated the iron overload-induced decrease in neuronal cell viability. This neuroprotective effect of HupA occurred concurrently with a decrease in ROS and an increase in ATP. Moreover, HupA treatment significantly blocked the upregulation of the LIP level and other aberrant iron metabolism changes induced by iron overload. Additionally, another specific AChE inhibitor, donepezil (Don), at a concentration that caused AChE inhibition equivalent to that of HupA negatively, influenced the aberrant changes in ROS, ATP or LIP that were induced by excessive iron. We provide the first demonstration of the protective effects of HupA against iron overload-induced neuronal damage. This beneficial role of HupA may be attributed to its attenuation of oxidative stress and mitochondrial dysfunction and elevation of LIP, and these effects are not associated with its AChE-inhibiting effect.

Acetylcholinesterase-independent protective effects of huperzine A against iron overload-induced oxidative damage and aberrant iron metabolism signaling in rat cortical neurons

PubMed Central

Tao, Ling-xue; Huang, Xiao-tian; Chen, Yu-ting; Tang, Xi-can; Zhang, Hai-yan

2016-01-01

Aim: Iron dyshomeostasis is one of the primary causes of neuronal death in Alzheimer's disease (AD). Huperzine A (HupA), a natural inhibitor of acetylcholinesterase (AChE), is a licensed anti-AD drug in China and a nutraceutical in the United Sates. Here, we investigated the protective effects of HupA against iron overload-induced injury in neurons. Methods: Rat cortical neurons were treated with ferric ammonium citrate (FAC), and cell viability was assessed with MTT assays. Reactive oxygen species (ROS) assays and adenosine triphosphate (ATP) assays were performed to assess mitochondrial function. The labile iron pool (LIP) level, cytosolic-aconitase (c-aconitase) activity and iron uptake protein expression were measured to determine iron metabolism changes. The modified Ellman's method was used to evaluate AChE activity. Results: HupA significantly attenuated the iron overload-induced decrease in neuronal cell viability. This neuroprotective effect of HupA occurred concurrently with a decrease in ROS and an increase in ATP. Moreover, HupA treatment significantly blocked the upregulation of the LIP level and other aberrant iron metabolism changes induced by iron overload. Additionally, another specific AChE inhibitor, donepezil (Don), at a concentration that caused AChE inhibition equivalent to that of HupA negatively, influenced the aberrant changes in ROS, ATP or LIP that were induced by excessive iron. Conclusion: We provide the first demonstration of the protective effects of HupA against iron overload-induced neuronal damage. This beneficial role of HupA may be attributed to its attenuation of oxidative stress and mitochondrial dysfunction and elevation of LIP, and these effects are not associated with its AChE-inhibiting effect. PMID:27498774

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

PubMed

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

2003-05-01

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

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.

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

Gustafsson, Helena; Runesson, Johan; Lundqvist, Jessica

The objective of the EU-funded integrated project ACuteTox is to develop a strategy in which general cytotoxicity, together with organ-specific toxicity and biokinetic features, are used for the estimation of human acute systemic toxicity. Our role in the project is to characterise the effect of reference chemicals with regard to neurotoxicity. We studied cell membrane potential (CMP), noradrenalin (NA) uptake, acetylcholine esterase (AChE) activity, acetylcholine receptor (AChR) signalling and voltage-operated calcium channel (VOCC) function in human neuroblastoma SH-SY5Y cells after exposure to 23 pharmaceuticals, pesticides or industrial chemicals. Neurotoxic alert chemicals were identified by comparing the obtained data with cytotoxicitymore » data from the neutral red uptake assay in 3T3 mouse fibroblasts. Furthermore, neurotoxic concentrations were correlated with estimated human lethal blood concentrations (LC50). The CMP assay was the most sensitive assay, identifying eight chemicals as neurotoxic alerts and improving the LC50 correlation for nicotine, lindane, atropine and methadone. The NA uptake assay identified five neurotoxic alert chemicals and improved the LC50 correlation for atropine, diazepam, verapamil and methadone. The AChE, AChR and VOCC assays showed limited potential for detection of acute toxicity. The CMP assay was further evaluated by testing 36 additional reference chemicals. Five neurotoxic alert chemicals were generated and orphendrine and amitriptyline showed improved LC50 correlation. Due to the high sensitivity and the simplicity of the test protocol, the CMP assay constitutes a good candidate assay to be included in an in vitro test strategy for prediction of acute systemic toxicity.« less

Chitooligosaccharides suppress the level of protein expression and acetylcholinesterase activity induced by Abeta25-35 in PC12 cells.

PubMed

Lee, Sang-Hoon; Park, Jin-Sook; Kim, Se-Kwon; Ahn, Chang-Bum; Je, Jae-Young

2009-02-01

Clinical applications of acetylcholinesterase (AChE) inhibitors are widespread in Alzheimer's sufferers in order to activate central cholinergic system and alleviate cognitive deficits by inhibiting the hydrolysis of acetylcholine. In this study, six kinds of chitooligosaccharides (COSs) with different molecular weight and degree of deacetylation were examined for their inhibitory effects against AChE. The 90-COSs exhibited potent AChE inhibitory activities compared to 50-COSs, while 90-MMWCOS (1000-5000 Da) in the 90-COSs showed the highest activity. Cell culture experiment revealed that 90-MMWCOS suppressed the level of AChE protein expression and AChE activity induced by Abeta(25-35) in PC12 cell lines.

[Effects of cornel iridoid glycoside on activity of cholinesterases in vitro].

PubMed

Chu, Si-Juan; Zhang, Lan; Liu, Gang; Zhou, Wen-Xia; Li, Lin

2013-05-01

The purpose of the present study was to investigate the effects of cornel iridoid glycoside (CIG) on the activity of cholinesterases in vitro, and to investigate the mechanism of CIG's treating Alzheimer's disease (AD). The sources of cholinesterases were prepared from human blood cells, rat brain homogenate and human blood plasma, respectively. The biochemical methods were used to detect the activity of acetylcholine esterase (AChE) and butyryl cholinesterase (BuChE) to investigate the influence of CIG on cholinesterases. The results showed that CIG inhibited the activity of AChE of human blood cells and rat brain homogenate, with the 50% inhibition rate (IC50) of 1.6 g . L-1 and 3.3 g . L-1, respectively; and the inhibition of AChE of CIG is reversible. CIG also inhibited the activity of BuChE of human blood plasma, with the IC50 of 2.9 g . L-1. In conclusion, CIG can inhibit the activity of AChE and BuChE in vitro, which may be one of the mechanisms of CIG to treat AD.

The lymphocytic cholinergic system and its contribution to the regulation of immune activity.

PubMed

Kawashima, Koichiro; Fujii, Takeshi

2003-12-26

Lymphocytes express most of the cholinergic components found in the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase. Stimulation of T and B cells with ACh or another mAChR agonist elicits intracellular Ca2+ signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and IL-2-induced signal transduction, probably via M3 and M5 mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Activation of T cells with phytohemagglutinin or antibodies against cell surface molecules enhances lymphocytic cholinergic transmission by activating expression of ChAT and M5 mAChR, which is suggestive of local cholinergic regulation of immune system activity. This idea is supported by the facts that lymphocytic cholinergic activity reflects well the changes in immune system function seen in animal models of immune deficiency and immune acceleration. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function.

Auger electron spectroscopy, secondary ion mass spectroscopy and optical characterization of a-C-H and BN films

NASA Technical Reports Server (NTRS)

Pouch, J. J.; Alterovitz, S. A.; Warner, J. D.

1986-01-01

The amorphous dielectrics a-C:H and BN were deposited on III-V semiconductors. Optical band gaps as high as 3 eV were measured for a-C:H generated by C4H10 plasmas; a comparison was made with bad gaps obtained from films prepared by CH4 glow discharges. The ion beam deposited BN films exhibited amorphous behavior with band gaps on the order of 5 eV. Film compositions were studied by Auger electron spectroscopy (AES), x-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). The optical properties were characterized by ellipsometry, UV/VIS absorption, and IR reflection and transmission. Etching rates of a-C:H subjected to O2 dicharges were determined.

Biocontrol of blue mold on apple fruits by Aureobasidium pullulans (strain Ach 1-1): in vitro and in situ evidence for the possible involvement of competition for nutrients.

PubMed

Bencheqroun, S Krimi; Bajji, M; Massart, S; Bentata, F; Labhilili, M; Achbani, H; El Jaafari, S; Jijakli, M H

2006-01-01

Aureobasidium pullulans strain Ach1-1 was recently isolated for its biocontrol effectiveness against Penicillium expansum, the causal agent of blue mold on harvested apples. In the present study, strain Ach1-1 was found to be very effective in controlling P. expansum on apple wounds. For in vitro tests, strain Ach1-1 and P. expansum were cocultured in the presence of apple juice (0 - 5%) using a system preventing direct contact between both agents. The presence of the antagonist greatly reduced germination of conidia at low (0.1, 0.5 and 1%) but not at high (5%) juice concentrations. Germination of previously inhibited conidia at 0.5% apple juice was partially restored in the presence of the antagonist when fresh juice was added at a final concentration of 5%, and completely recovered at both 0.5 and 5% juice concentrations in the absence of the antagonist. These data show that P. expansum conidia are able to germinate when cocultered with strain Ach1-1 in conditions of sufficient rather than limited nutrient availability and that the antagonist does not affect the viability of these conidia, indicating that the inhibitory effect of strain Ach1-1 on conidia germination may be due to a competition for nutrients. Such observation was confirmed in situ since the application of high amounts of exogenous amino acids, vitamins or sugars on apple wounds significantly reduced the protective level of strain Ach1-1 against P. expansum, the most important effect was obtained with amino acids followed by vitamins and then by sugars. The present work provides both in vitro and in situ evidence that the biocontrol activity of strain Ach1-1 against P. expansum essentially relies on competition for apple fruit nutrients, especially amino acids.

Nicotine/Cigarette-smoke Promotes Metastasis of Pancreatic Cancer Through α7nAChR-mediated MUC4 Up-regulation

PubMed Central

Momi, Navneet; Ponnusamy, Moorthy P.; Kaur, Sukhwinder; Rachagani, Satyanarayana; Kunigal, Sateesh S; Chellappan, Srikumar; Ouellette, Michel M; Batra, Surinder K

2012-01-01

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well-established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared to the normal pancreas and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette-smoke-extract (CSE) and nicotine, which is the major component of CS, significantly up-regulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. Additionally, we demonstrated that nicotine-mediated MUC4 up-regulation promotes the PC cell migration through the activation of the downstream effectors such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in-vivo studies demonstrated a dramatic increase in the mean pancreatic tumor weight [low-dose (100 mg/m3 TSP), p=0.014; high-dose (247 mg/m3 TSP), p=0.02] and significant tumor metastasis to various distant organs in the CS-exposed-mice, orthotopically implanted with luciferase-transfected PC cells, as compared to the sham-controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine [low-dose, 155.88±35.96 ng/ml; high-dose, 216.25±29.95 ng/ml] and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings revealed for the first time that CS up

Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery.

PubMed

Simm, Jaak; Klambauer, Günter; Arany, Adam; Steijaert, Marvin; Wegner, Jörg Kurt; Gustin, Emmanuel; Chupakhin, Vladimir; Chong, Yolanda T; Vialard, Jorge; Buijnsters, Peter; Velter, Ingrid; Vapirev, Alexander; Singh, Shantanu; Carpenter, Anne E; Wuyts, Roel; Hochreiter, Sepp; Moreau, Yves; Ceulemans, Hugo

2018-05-17

In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

Biallelic mutation of UNC50, encoding a protein involved in AChR trafficking, is responsible for arthrogryposis.

PubMed

Abiusi, Emanuela; D'Alessandro, Manuela; Dieterich, Klaus; Quevarec, Loic; Turczynski, Sandrina; Valfort, Aurore-Cecile; Mezin, Paulette; Jouk, Pierre Simon; Gut, Marta; Gut, Ivo; Bessereau, Jean Louis; Melki, Judith

2017-10-15

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The Ache: Genocide Continues in Paraguay. IWGIA Document No. 17.

ERIC Educational Resources Information Center

Munzel, Mark

In 1972, the Paraguayan Roman Catholic Church protested against the massacre of Indians in Paraguay. This was followed by further protests from Paraguayan intellectuals. These protests led to the removal of Jesus de Pereira, one of the executors of the official Ache policy. Thus, the critics were appeased. Since the beginning of 1973, new protests…

Determining Antioxidant Activities of Lactobacilli Cell-Free Supernatants by Cellular Antioxidant Assay: A Comparison with Traditional Methods

PubMed Central

Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q.; Chen, Wei

2015-01-01

Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs. PMID:25789875

Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

PubMed Central

Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

1999-01-01

Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10464070

New generation QuIC assays for prion seeding activity.

PubMed

Orrù, Christina D; Wilham, Jason M; Vascellari, Sarah; Hughson, Andrew G; Caughey, Byron

2012-01-01

The ability of abnormal TSE-associated forms of PrP to seed the formation of amyloid fibrils from recombinant PrP(Sen) has served as the basis for several relatively rapid and highly sensitive tests for prion diseases. These tests include rPrP-PMCA (rPMCA), standard quaking-induced conversion (S-QuIC), amyloid seeding assay (ASA), real-time QuIC (RT-QuIC) and enhanced QuIC (eQuIC). Here, we summarize recent improvements in the RT-QuIC-based assays that enhance the practicality, sensitivity and quantitative attributes of assays QuIC and promote the detection of prion seeding activity in dilute, inhibitor-laden fluids such as blood plasma.

A Sensitive and Versatile Fluorescent Activity Assay for ABHD6.

PubMed

Savinainen, Juha R; Navia-Paldanius, Dina; Laitinen, Jarmo T

2016-01-01

The α/β-hydrolase domain-containing 6 (ABHD6) enzyme is a newly found serine hydrolase whose substrate profile resembles that of monoacylglycerol lipase (MAGL), the major 2-arachidonoyl glycerol (2-AG) hydrolase in the brain. Here, we describe a sensitive fluorescent assay of ABHD6 activity in a 96-well-plate format that allows parallel testing of inhibitor activities of up to 40 compounds in a single assay. The method utilizes lysates of HEK293 cells transiently overexpressing human ABHD6 as the enzymatic source, and kinetically monitors glycerol liberated in the hydrolysis of 1(3)-AG, the preferred arachidonoyl glycerol isomer. Glycerol output is coupled to an enzymatic cascade generating the fluorescent end-product resorufin. The approach has major benefits compared to laborious traditional mass spectrometric methods and liquid scintillation-based assays, or approaches using unnatural substrates.

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.

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

PubMed

Bagrov, Ia Iu; Manusova, N B

2011-01-01

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

Analysis of the activation of acetylcholinesterase by carbon nanoparticles using a monolithic immobilized enzyme microreactor: role of the water molecules in the active site gorge.

PubMed

Ibrahim, Firas; Andre, Claire; Iutzeler, Anne; Guillaume, Yves Claude

2013-10-01

A biochromatographic system was used to study the direct effect of carbon nanoparticles (CNPs) on the acetylcholinesterase (AChE) activity. The AChE enzyme was covalently immobilized on a monolithic CIM-disk via its NH2 residues. Our results showed an increase in the AChE activity in presence of CNPs. The catalytic constant (k(cat)) was increased while the Michaelis constant (K(m)) was slightly decreased. This indicated an increase in the enzyme efficiency with increase of the substrate affinity to the active site. The thermodynamic data of the activation mechanism of the enzyme, i.e. ΔH* and ΔS*, showed no change in the substrate interaction mechanism with the anionic binding site. The increase of the enthalpy (ΔH*) and the entropy (ΔS*) with decrease in the free energy of activation (Ea) was related to structural conformation change in the active site gorge. This affected the stability of water molecules in the active site gorge and facilitated water displacement by substrate for entering to the active site of the enzyme.

Anti-listeria activity of poly(lactic acid)/sawdust particle biocomposite film impregnated with pediocin PA-1/AcH and its use in raw sliced pork.

PubMed

Woraprayote, Weerapong; Kingcha, Yutthana; Amonphanpokin, Pannawit; Kruenate, Jittiporn; Zendo, Takeshi; Sonomoto, Kenji; Benjakul, Soottawat; Visessanguan, Wonnop

2013-10-15

A novel poly(lactic acid) (PLA)/sawdust particle (SP) biocomposite film with anti-listeria activity was developed by incorporation of pediocin PA-1/AcH (Ped) using diffusion coating method. Sawdust particle played an important role in embedding pediocin into the hydrophobic PLA film. The anti-listeria activity of the PLA/SP biocomposite film incorporated with Ped (PLA/SP+Ped) was detected, while no activity against the tested pathogen was observed for the control PLA films (without SP and/or Ped). Dry-heat treatment of film before coating with Ped resulted in the highest Ped adsorption (11.63 ± 3.07 μg protein/cm(2)) and the highest anti-listeria activity. A model study of PLA/SP+Ped as a food-contact antimicrobial packaging on raw sliced pork suggests a potential inhibition of Listeria monocytogenes (99% of total listerial population) on raw sliced pork during the chilled storage. This study supports the feasibility of using PLA/SP+Ped film to reduce the initial load of L. monocytogenes on the surface of raw pork. © 2013.

α7 Nicotinic Acetylcholine Receptor (α7nAChR) Expression in Bone Marrow–Derived Non–T Cells Is Required for the Inflammatory Reflex

PubMed Central

Olofsson, Peder S; Katz, David A; Rosas-Ballina, Mauricio; Levine, Yaakov A; Ochani, Mahendar; Valdés-Ferrer, Sergio I; Pavlov, Valentin A; Tracey, Kevin J; Chavan, Sangeeta S

2012-01-01

The immune response to infection or injury coordinates host defense and tissue repair, but also has the capacity to damage host tissues. Recent advances in understanding protective mechanisms have found neural circuits that suppress release of damaging cytokines. Stimulation of the vagus nerve protects from excessive cytokine production and ameliorates experimental inflammatory disease. This mechanism, the inflammatory reflex, requires the α7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel expressed on macrophages, lymphocytes, neurons and other cells. To investigate cell-specific function of α7nAChR in the inflammatory reflex, we created chimeric mice by cross-transferring bone marrow between wild-type (WT) and α7nAChR-deficient mice. Deficiency of α7nAChR in bone marrow–derived cells significantly impaired vagus nerve–mediated regulation of tumor necrosis factor (TNF), whereas α7nAChR deficiency in neurons and other cells had no significant effect. In agreement with recent work, the inflammatory reflex was not functional in nude mice, because functional T cells are required for the integrity of the pathway. To investigate the role of T-cell α7nAChR, we adoptively transferred α7nAChR-deficient or WT T cells to nude mice. Transfer of WT and α7nAChR-deficient T cells restored function, indicating that α7nAChR expression on T cells is not necessary for this pathway. Together, these results indicate that α7nAChR expression in bone marrow–derived non–T cells is required for the integrity of the inflammatory reflex. PMID:22183893

The binding properties of cycloxaprid on insect native nAChRs partially explain the low cross-resistance with imidacloprid in Nilaparvata lugens.

PubMed

Zhang, Yixi; Xu, Xiaoyong; Bao, Haibo; Shao, Xusheng; Li, Zhong; Liu, Zewen

2018-06-06

Neonicotinoids, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) to control Nilaparvata lugens, a major rice insect pest. High imidacloprid resistance has been reported in N. lugens in laboratory and in fields. Cycloxaprid, an oxabridged cis-nitromethylene neonicotinoid, showed high insecticidal activity against N. lugens and low cross-resistance in the imidacloprid resistant strains and field populations. Binding studies have demonstrated that imidacloprid had two binding sites with different affinities (Kd = 3.18 ± 0.43 pM and 1.78 ± 0.19 nM) in N. lugens nAChRs. Cycloxaprid was poor at displacing [ 3 H]imidacloprid at its high-affinity binding site (Ki = 159.38±20.43 nM), but quite efficient at the low-affinity binding site (Ki = 1.27±0.35 nM). These data showed that cycloxaprid had overlapping binding sites with imidacloprid only at its low-affinity binding site. Therefore, the low displacement ability of cycloxaprid against imidacloprid binding at its high affinity site could partially explain the low cross-resistance of cycloxaprid in the imidacloprid resistant populations. The high insecticidal activity, low cross-resistance and different binding properties on insect nAChRs of cycloxaprid demonstrating it a potential insecticide to control N. lugens and related insect pests, especially the ones with high resistance to neonicotinoids. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

PubMed

Nagatsu, T; Oka, K; Kato, T

1979-07-21

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

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.

A new effective assay to detect antimicrobial activity of filamentous fungi.

PubMed

Pereira, Eric; Santos, Ana; Reis, Francisca; Tavares, Rui M; Baptista, Paula; Lino-Neto, Teresa; Almeida-Aguiar, Cristina

2013-01-15

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time. Copyright © 2012 Elsevier GmbH. All rights reserved.

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.

Cracking the Betel Nut: Cholinergic Activity of Areca Alkaloids and Related Compounds.

PubMed

Horenstein, Nicole A; Quadri, Marta; Stokes, Clare; Shoaib, Mohammed; Papke, Roger L

2017-10-03

The use of betel quid is the most understudied major addiction in the world. The neuropsychological activity of betel quid has been attributed to alkaloids of Areca catechu. With the goal of developing novel addiction treatments, we evaluate the muscarinic and nicotinic activity of the four major Areca alkaloids: arecoline, arecaidine, guvacoline, and guvacine and four structurally related compounds. Acetylcholine receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Both arecoline- and guvacoline-activated muscarinic acetylcholine receptors (mAChR), while only arecoline produced significant activation of nicotinic AChR (nAChR). We characterized four additional arecoline-related compounds, seeking an analog that would retain selective activity for a α4* nAChR, with diminished effects on mAChR and not be a desensitizer of α7 nAChR. We show that this profile is largely met by isoarecolone. Three additional arecoline analogs were characterized. While the quaternary dimethyl analog had a broad range of activities, including activation of mAChR and muscle-type nAChR, the methyl analog only activated a range of α4* nAChR, albeit with low potency. The ethyl analog had no detectable cholinergic activity. Evidence indicates that α4* nAChR are at the root of nicotine addiction, and this may also be the case for betel addiction. Our characterization of isoarecolone and 1-(4-methylpiperazin-1-yl) ethanone as truly selective α4*nAChR selective partial agonists with low muscarinic activity may point toward a promising new direction for the development of drugs to treat both nicotine and betel addiction. Nearly 600 million people use Areca nut, often with tobacco. Two of the Areca alkaloids are muscarinic acetylcholine receptor agonists, and one, arecoline, is a partial agonist for the α4* nicotinic acetylcholine receptors (nAChR) associated with tobacco addiction. The profile of arecoline activity suggested its potential to be used as a

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.

Is fast fiber innervation responsible for increased acetylcholinesterase activity in reinnervating soleus muscles?

NASA Technical Reports Server (NTRS)

Misulis, K. E.; Dettbarn, W. D.

1985-01-01

An investigation was conducted as to whether the predominantly slow SOL, which is low in AChE activity, is initially reinnervated by axons that originally innervated fast muscle fibers with high AChE activity, such as those of the EDL. Local denervation of the SOL in the guinea pig was performed because this muscle is composed solely of slow (type I) fibers; thereby virtually eliminating the possibility of homologous muscle fast fiber innervation. The overshoot in this preparation was qualitatively similar to that seen with distal denervation in the guinea pig and local and distal denervation in the rat. Thus, initial fast fiber innvervation is not responsible for the patterns of change in AChE activity seen with reinnervation in the SOL. It is concluded that the neural control of AChe is different in these two muscles and may reflect specific differences in the characteristics of AChE regulation in fast and slow muscle.

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.

A novel 96-well gel-based assay for determining antifungal activity against filamentous fungi.

PubMed

Troskie, Anscha Mari; Vlok, Nicolas Maré; Rautenbach, Marina

2012-12-01

In recent years the global rise in antibiotic resistance and environmental consciousness lead to a renewed fervour to find and develop novel antibiotics, including antifungals. However, the influence of the environment on antifungal activity is often disregarded and many in vitro assays may cause the activity of certain antifungals to be overestimated or underestimated. The general antifungal test assays that are economically accessible to the majority of scientists primarily rely on visual examination or on spectrophotometric analysis. The effect of certain morphogenic antifungals, which may lead to hyperbranching of filamentous fungi, unfortunately renders these methods unreliable. To minimise the difficulties experienced as a result of hyperbranching, we developed a straightforward, economical 96-well gel-based method, independent of spectrophotometric analysis, for highly repeatable determination of antifungal activity. For the calculation of inhibition parameters, this method relies on the visualisation of assay results by digitisation. The antifungal activity results from our novel micro-gel dilution assay are comparable to that of the micro-broth dilution assay used as standard reference test of The Clinical and Laboratory Standard Institute. Furthermore, our economical assay is multifunctional as it permits microscopic analysis of the preserved assay results, as well as rendering highly reliable data. Copyright © 2012 Elsevier B.V. All rights reserved.

Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements.

PubMed

Wolfe, Kelly L; Liu, Rui Hai

2007-10-31

A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

Functional characterization of α7 nicotinic acetylcholine and NMDA receptor signaling in SH-SY5Y neuroblastoma cells in an ERK phosphorylation assay.

PubMed

Elnagar, Mohamed R; Walls, Anne Byriel; Helal, Gouda K; Hamada, Farid M; Thomsen, Morten Skøtt; Jensen, Anders A

2018-05-05

In the present study, the functional properties of α7 nicotinic acetylcholine receptors (α7 nAChRs) and N-methyl-D-aspartate receptors (NMDARs) endogenously expressed in SH-SY5Y human neuroblastoma cells were characterized in an extracellular-signal regulated kinase (ERK) phosphorylation assay. Both choline and N-methyl-D-aspartate (NMDA) mediated robust concentration-dependent increases in ERK phosphorylation in the SH-SY5Y cells, exhibiting EC 50 values in good agreement with those reported for the agonists at recombinant α7 nAChRs and NMDARs, respectively. Importantly, the responses evoked by choline (10 mM) and by NMDA (50 μM) were significantly inhibited by the α7-selective antagonist α-bungarotoxin (100 nM) and by the NMDAR-selective antagonist MK-801 (50 μM), respectively. The increased ERK phosphorylation levels observed upon co-application of choline (1, 3, 10 mM) and NMDA (50 μM) compared to those produced by the two agonists on their own were fully reconcilable with additive effects and did not reveal substantial synergy between α7 nAChR and NMDAR signaling. Interestingly, however, the responses evoked by the "choline (10 mM) - NMDA (50 μM)" combination were almost completely inhibited by α-bungarotoxin (100 nM) as well as by MK-801 (50 μM), suggesting some sort of a link between α7 nAChR- and NMDAR-mediated ERK phosphorylation. Finally, oligomeric amyloid-β 1-42 peptide (1000 nM) mediated robust inhibition of the ERK phosphorylation induced by choline (10 mM), NMDA (50 μM) and the "choline (10 mM) - NMDA (50 μM)" combination. In conclusion, ERK phosphorylation measurements in SH-SY5Y cells provides a robust assay for studies of α7 nAChR- and NMDAR-mediating signaling and putative functional interactions between the receptors. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis and anticholinesterase activity of coumarin-3-carboxamides bearing tryptamine moiety.

PubMed

Ghanei-Nasab, Samaneh; Khoobi, Mehdi; Hadizadeh, Farzin; Marjani, Azam; Moradi, Alireza; Nadri, Hamid; Emami, Saeed; Foroumadi, Alireza; Shafiee, Abbas

2016-10-04

A number of N-(2-(1H-indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamides were synthesized and tested against AChE and BuChE. The in vitro assessment of the synthesized compounds 4a-o revealed that most of them had significant activity toward AChE. The SAR study demonstrated that the introduction of benzyloxy moiety on the 7-position of coumarin scaffold can improve the anti-AChE activity. The best result was obtained with 7-(4-fluorobenzyl)oxy moiety in the case of compound 4o, displaying IC50 value of 0.16 μM. Based on the docking study of AChE, the prototype compound 4o was laid across the active site and occupied both peripheral anionic site (PAS) and catalytic anionic site (CAS). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Midwifery students attribute different quantitative meanings to hurt, ache and pain: a cross-sectional survey.

PubMed

Bergh, Ingrid H E; Ek, Kristina; Mårtensson, Lena B

2013-06-01

Assessment of women's labor pain is seldom acknowledged in clinical practice or research. The words "aching" and "hurting" are frequently used by women to describe childbirth pain. The aim of this study was to determine the quantitative meanings midwifery students attribute to the terms "hurt", "ache" and "pain". Data was collected by self-administered questionnaire from students at seven Swedish midwifery programs. A total of 230 filled out and returned a completed questionnaire requesting them to rate, on a visual analog scale, the intensity of "hurt", "ache" or "pain" in the back, as reported by a fictitious parturient. The midwifery students attributed, with substantial individual variation, different quantitative meanings to the studied pain descriptors. To be able to communicate about pain with a woman in labor, it is essential that the midwife be familiar with the value of different words and what they mean to her as this may affect her assessment when the woman describes her pain. Copyright © 2012 Australian College of Midwives. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-11-01

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

Antioxidant activity of puha (Sonchus oleraceus L.) as assessed by the cellular antioxidant activity (CAA) assay.

PubMed

McDowell, Arlene; Thompson, Scott; Stark, Mirjam; Ou, Zong-Quan; Gould, Kevin S

2011-12-01

There is considerable interest in antioxidant dietary components that can be protective against degenerative diseases in humans. Puha (Sonchus oleraceus L.) is a rich source of polyphenols, and exhibits strong antioxidant activity as measured by the 2,2-diphenylpicrylhydrazyl (DPPH) assay. However, the potential of puha to protect against degenerative diseases requires that low molecular weight antioxidants (LMWA) are absorbed by, and active in, human cells. The cellular antioxidant activity (CAA) assay was used to investigate the antioxidant activity of puha leaf extracts. Preparation methods of freezing and freeze-drying reduced the total polyphenolic content compared with fresh puha, but did not affect the LMWA potential as determined by the DPPH assay. The IC(50) values were 0.012 ± 0.003 mg/mL and 0.010 ± 0.005 mg/mL for freeze-dried and fresh puha leaves, respectively. Using the CAA assay, it was shown that LMWAs from foliar extracts of puha were effectively absorbed into HepG2 cells, and exerted antioxidant activity at levels comparable to those of extracts from blueberry fruits, the much-touted antioxidant superfood. Methylene blue staining of HepG2 cells indicated that puha extracts were not cytotoxic at concentrations below 100 mg DW/mL. The data indicate the potential of puha as a nutraceutical supplement for human health. Copyright © 2011 John Wiley & Sons, Ltd.

Anticholinesterase and Antityrosinase Activities of Ten Piper Species from Malaysia

PubMed Central

Salleh, Wan Mohd Nuzul Hakimi Wan; Hashim, Nur Athirah; Ahmad, Farediah; Heng Yen, Khong

2014-01-01

Purpose: The aim of this study was to investigate acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and antityrosinase activities of extracts from ten Piper species namely; P. caninum, P. lanatum, P. abbreviatum, P. aborescens, P. porphyrophyllum, P. erecticaule, P. ribesioides, P. miniatum, P. stylosum, and P. majusculum. Methods: Anticholinesterase and antityrosinase activities were evaluated against in vitro Ellman spectroscopy method and mushroom tyrosinase, respectively. Results: The EtOAc extract of P. erecticaule showed the highest AChE and BChE inhibitory with 22.9% and 70.9% inhibition, respectively. In antityrosinase activity, all extracts of P. porphyrophyllum showed the highest inhibitory effects against mushroom tyrosinase, compared to standard, kojic acid. Conclusion: This study showed that P. erecticaule and P. porphyrophyllum have potential AChE/BChE and tyrosinase inhibition activities. The respective extracts can be explored further for the development of novel lead as AChE/BChE and tyrosinase inhibitors in therapeutic management of Alzheimer’s disease. PMID:25671185

Exposure to acetylcholinesterase inhibitors alters the physiology and motor function of honeybees.

PubMed

Williamson, Sally M; Moffat, Christopher; Gomersall, Martha A E; Saranzewa, Nastja; Connolly, Christopher N; Wright, Geraldine A

2013-01-01

Cholinergic signaling is fundamental to neuromuscular function in most organisms. Sub-lethal doses of neurotoxic pesticides that target cholinergic signaling can alter the behavior of insects in subtle ways; their influence on non-target organisms may not be readily apparent in simple mortality studies. Beneficial arthropods such as honeybees perform sophisticated behavioral sequences during foraging that, if influenced by pesticides, could impair foraging success and reduce colony health. Here, we investigate the behavioral effects on honeybees of exposure to a selection of pesticides that target cholinergic signaling by inhibiting acetylcholinesterase (AChE). To examine how continued exposure to AChE inhibitors affected motor function, we fed adult foraging worker honeybees sub-lethal concentrations of these compounds in sucrose solution for 24 h. Using an assay for locomotion in bees, we scored walking, stopped, grooming, and upside down behavior continuously for 15 min. At a 10 nM concentration, all the AChE inhibitors caused similar effects on behavior, notably increased grooming activity and changes in the frequency of bouts of behavior such as head grooming. Coumaphos caused dose-dependent effects on locomotion as well as grooming behavior, and a 1 μM concentration of coumaphos induced symptoms of malaise such as abdomen grooming and defecation. Biochemical assays confirmed that the four compounds we assayed (coumaphos, aldicarb, chlorpyrifos, and donepezil) or their metabolites acted as AChE inhibitors in bees. Furthermore, we show that transcript expression levels of two honeybee AChE inhibitors were selectively upregulated in the brain and in gut tissues in response to AChE inhibitor exposure. The results of our study imply that the effects of pesticides that rely on this mode of action have subtle yet profound effects on physiological effects on behavior that could lead to reduced survival.

Exposure to Acetylcholinesterase Inhibitors Alters the Physiology and Motor Function of Honeybees

PubMed Central

Williamson, Sally M.; Moffat, Christopher; Gomersall, Martha A. E.; Saranzewa, Nastja; Connolly, Christopher N.; Wright, Geraldine A.

2013-01-01

Cholinergic signaling is fundamental to neuromuscular function in most organisms. Sub-lethal doses of neurotoxic pesticides that target cholinergic signaling can alter the behavior of insects in subtle ways; their influence on non-target organisms may not be readily apparent in simple mortality studies. Beneficial arthropods such as honeybees perform sophisticated behavioral sequences during foraging that, if influenced by pesticides, could impair foraging success and reduce colony health. Here, we investigate the behavioral effects on honeybees of exposure to a selection of pesticides that target cholinergic signaling by inhibiting acetylcholinesterase (AChE). To examine how continued exposure to AChE inhibitors affected motor function, we fed adult foraging worker honeybees sub-lethal concentrations of these compounds in sucrose solution for 24 h. Using an assay for locomotion in bees, we scored walking, stopped, grooming, and upside down behavior continuously for 15 min. At a 10 nM concentration, all the AChE inhibitors caused similar effects on behavior, notably increased grooming activity and changes in the frequency of bouts of behavior such as head grooming. Coumaphos caused dose-dependent effects on locomotion as well as grooming behavior, and a 1 μM concentration of coumaphos induced symptoms of malaise such as abdomen grooming and defecation. Biochemical assays confirmed that the four compounds we assayed (coumaphos, aldicarb, chlorpyrifos, and donepezil) or their metabolites acted as AChE inhibitors in bees. Furthermore, we show that transcript expression levels of two honeybee AChE inhibitors were selectively upregulated in the brain and in gut tissues in response to AChE inhibitor exposure. The results of our study imply that the effects of pesticides that rely on this mode of action have subtle yet profound effects on physiological effects on behavior that could lead to reduced survival. PMID:23386834

Specific radioisotopic assay for cholinesterase. Technical report, December 1987-March 1988

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

Talbot, B.G.; Anderson, D.R.; Harris, L.W.

1990-05-01

The radiometric method (I) for measuring ChE activity was modified to preclude the use of p-dioxane, a hazardous material. The modified procedure (II) uses 0.4 N perchloric acid (PCA), instead of p-dioxane, to denature the ChE and stop hydrolysis of 14C-acetylcholine (ACh). The unreacted substrate (ACh) is removed by cationic exchange resin suspended in water. The supernatant (acidic water solution) containing the product of hydrolysis, 14C-acetic acid, is mixed with nonhazardous scintillation cocktail and counted. The incubation mixture (37 degrees C) for II is similar to I and is composed of 0.1 ml of buffer-salt solution (pH 7.8), 0.1 mlmore » of guinea pig whole blood (WB)-water suspension and 0.1 ml of 3mM ACh solution.« less

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

PubMed

Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

2018-06-05

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

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

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

DOE PAGES

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.; ...

2018-05-14

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

Cell based assays for anti-Plasmodium activity evaluation.

PubMed

Mokgethi-Morule, Thabang; N'Da, David D

2016-03-10

Malaria remains one of the most common and deadly infectious diseases worldwide. The severity of this global public health challenge is reflected by the approximately 198 million people, who were reportedly infected in 2013 and by the more than 584,000 related deaths in that same year. The rising emergence of drug resistance towards the once effective artemisinin combination therapies (ACTs) has become a serious concern and warrants more robust drug development strategies, with the objective of eradicating malaria infections. The intricate biology and life cycle of Plasmodium parasites complicate the understanding of the disease in such a way that would enhance the development of more effective chemotherapies that would achieve radical clinical cure and that would prevent disease relapse. Phenotypic cell based assays have for long been a valuable approach and involve the screening and analysis of diverse compounds with regards to their activities towards whole Plasmodium parasites in vitro. To achieve the Millennium Development Goal (MDG) of malaria eradication by 2020, new generation drugs that are active against all parasite stages (erythrocytic (blood), exo-erythrocytic (liver stages and gametocytes)) are needed. Significant advances are being made in assay development to overcome some of the practical challenges of assessing drug efficacy, particularly in the liver and transmission stage Plasmodium models. This review discusses primary screening models and the fundamental progress being made in whole cell based efficacy screens of anti-malarial activity. Ongoing challenges and some opportunities for improvements in assay development that would assist in the discovery of effective, safe and affordable drugs for malaria treatments are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

In-vivo measurements of regional acetylcholine esterase activity in degenerative dementia: comparison with blood flow and glucose metabolism.

PubMed

Herholz, K; Bauer, B; Wienhard, K; Kracht, L; Mielke, R; Lenz, M O; Strotmann, T; Heiss, W D

2000-01-01

Memory and attention are cognitive functions that depend heavily on the cholinergic system. Local activity of acetylcholine esterase (AChE) is an indicator of its integrity. Using a recently developed tracer for positron emission tomography (PET), C-11-labeled N-methyl-4-piperidyl-acetate (C11-MP4A), we measured regional AChE activity in 4 non-demented subjects, 4 patients with dementia of Alzheimer type (DAT) and 1 patient with senile dementia of Lewy body type (SDLT), and compared the findings with measurements of blood flow (CBF) and glucose metabolism (CMRGlc). Initial tracer extraction was closely related to CBF. AChE activity was reduced significantly in all brain regions in demented subjects, whereas reduction of CMRGlc and CBF was more limited to temporo-parietal association areas. AChE activity in SDLT was in the lower range of values in DAT. Our results indicate that, compared to non-demented controls, there is a global reduction of cortical AChE activity in dementia. Dementia, cholinergic system, acetylcholine esterase, positron emission tomography, cerebral blood flow, cerebral glucose metabolism.

nAChRs-ERK1/2-Egr-1 signaling participates in the developmental toxicity of nicotine by epigenetically down-regulating placental 11β-HSD2.

PubMed

Zhou, Jin; Liu, Fulin; Yu, Luting; Xu, Dan; Li, Bin; Zhang, Guohui; Huang, Wen; Li, Lu; Zhang, Yuanzhen; Zhang, Wei; Wang, Hui

2018-04-01

Impaired placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity which inactivates maternal glucocorticoids is associated with poor fetal growth and a higher risk of chronic diseases in adulthood. This study aimed to elucidate the epigenetically regulatory mechanism of nicotine on placental 11β-HSD2 expression. Pregnant Wistar rats were administered 1.0 mg/kg nicotine subcutaneously twice a day from gestational day 9 to 20. The results showed that prenatal nicotine exposure increased corticosterone levels in the placenta and fetal serum, disrupted placental morphology and endocrine function, and reduced fetal bodyweight. Meanwhile, histone modification abnormalities (decreased acetylation and increased di-methylation of histone 3 Lysine 9) on the HSD11B2 promoter and lower-expression of 11β-HSD2 were observed. Furthermore, the expression of nicotinic acetylcholine receptor (nAChR) α4/β2, the phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and Ets-like protein-1 (Elk-1), and the expression of early growth response-1 (Egr-1) were increased in the nicotine groups. In human BeWo cells, nicotine decreased 11β-HSD2 expression, increased nAChRα9 expression, and activated ERK1/2/Elk-1/Egr-1 signaling in the concentration (0.1-10 μM)-dependent manner. Antagonism of nAChRs, inhibition of ERK1/2 and Egr-1 knockdown by siRNA were able to block/abrogate the effects of nicotine on histone modification and expression of 11β-HSD2. Taken together, nicotine can impair placental structure and function, and induce fetal developmental toxicity. The underlying mechanism involves histone modifications and down-regulation of 11β-HSD2 through nAChRs/ERK1/2/Elk-1/Egr-1 signaling, which increases active glucocorticoids levels in the placenta and fetus, and eventually inhibits the fetal development. Copyright © 2018 Elsevier Inc. All rights reserved.

[Detection of viable metabolically active yeast cells using a colorimetric assay].

PubMed

Růzicka, F; Holá, V

2008-02-01

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

Acetylcholinesterase and butyrylcholinesterase inhibitory activity of Pinus species essential oils and their constituents.

PubMed

Bonesi, Marco; Menichini, Federica; Tundis, Rosa; Loizzo, Monica R; Conforti, Filomena; Passalacqua, Nicodemo G; Statti, Giancarlo A; Menichini, Francesco

2010-10-01

This study aimed to investigate the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity of the essential oils from Pinus nigra subsp. nigra, P. nigra var. calabrica, and P. heldreichii subsp. leucodermis. This activity is relevant to the treatment of Alzheimer's disease (AD), since cholinesterase drugs are currently the only drugs available to treat AD. P. heldreichii subsp. leucodermis exhibited the most promising activity, with IC(50) values of 51.1 and 80.6 microg/mL against AChE and BChE, respectively. An interesting activity against AChE was also observed with P. nigra subsp. nigra essential oil, with an IC(50) value of 94.4 microg/mL. Essential oils were analyzed by GC and GC-MS with the purpose of investigating their relationships with the observed activities. Among the identified constituents, terpinolene, beta-phellandrene, linalyl acetate, trans-caryophyllene, and terpinen-4-ol were tested. trans-Caryophyllene and terpinen-4-ol inhibited BChE with IC(50) values of 78.6 and 107.6 microg/mL, respectively. beta-Phellandrene was selective against AChE (IC(50) value of 120.2 microg/mL).

Protein-mimicking nanowire-inspired electro-catalytic biosensor for probing acetylcholinesterase activity and its inhibitors.

PubMed

Zhang, Qingqing; Hu, Yufang; Wu, Di; Ma, Shaohua; Wang, Jiao; Rao, Jiajia; Xu, Lihua; Xu, Huan; Shao, Huili; Guo, Zhiyong; Wang, Sui

2018-06-01

A highly sensitive electrochemical biosensor based on the synthetized L-Cysteine-Ag(I) coordination polymer (L-Cys-Ag(I) CP), which looks like a protein-mimicking nanowire, was constructed to detect acetylcholinesterase (AChE) activity and screen its inhibitors. This sensing strategy involves the reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce hydrogen peroxide (H 2 O 2 ), thus L-Cys-Ag(I) CP possesses the electro-catalytic property to H 2 O 2 reduction. Herein, the protein-mimicking nanowire-based platform was capable of investigating successive of H 2 O 2 effectively by amperometric i-t (current-time) response, and was further applied for the turn-on electrochemical detection of AChE activity. The proposed sensor is highly sensitive (limit of detection is 0.0006 U/L) and is feasible for screening inhibitors of AChE. The model for AChE inhibition was further established and two traditional AChE inhibitors (donepezil and tacrine) were employed to verify the feasibility of the system. The IC 5 0 of donepezil and tacrine were estimated to be 1.4 nM and 3.5 nM, respectively. The developed protocol provides a new and promising platform for probing AChE activity and screening its inhibitors with low cost, high sensitivity and selectivity. Copyright © 2018 Elsevier B.V. All rights reserved.

Nicotine withdrawal-induced inattention is absent in alpha7 nAChR knockout mice

PubMed Central

Higa, K. K.; Grim, A.; Kamenski, M. E.; van Enkhuizen, J.; Zhou, X.; Li, K.; Naviaux, J. C.; Wang, L.; Naviaux, R. K.; Geyer, M. A.; Markou, A.; Young, J. W.

2017-01-01

Rationale Smoking is the leading cause of preventable death in the U.S., but quit attempts result in withdrawal-induced cognitive dysfunction and predicts relapse. Greater understanding of the neural mechanism(s) underlying these cognitive deficits is required to develop targeted treatments to aid quit attempts. Objectives We examined nicotine withdrawal-induced inattention in mice lacking the α7 nicotinic acetylcholine receptor (nAChR) using the 5-choice continuous performance test (5C-CPT). Methods Mice were trained in the 5C-CPT prior to osmotic minipump implantation containing saline or nicotine. Experiment 1 used 40 mg/kg/day nicotine treatment and tested C57BL/6 mice 4, 28, and 52 h after pump removal. Experiment 2 used 14 and 40 mg/kg/day nicotine treatment in α7 nAChR knockout (KO) and wildtype (WT) littermates tested 4 h after pump removal. Subsets of WT mice were sacrificed before and after pump removal to assess changes in receptor expression associated with nicotine administration and withdrawal. Results Nicotine withdrawal impaired attention in the 5C-CPT, driven by response inhibition and target detection deficits. The overall attentional deficit was absent in α7 nAChR KO mice despite response disinhibition in these mice. Synaptosomal glutamate mGluR5 and dopamine D4 receptor expression were reduced during chronic nicotine but increased during withdrawal, potentially contributing to cognitive deficits. Conclusions The α7 nAChR may underlie nicotine withdrawal-induced deficits in target detection but is not required for response disinhibition deficits. Alterations to the glutamatergic and dopaminergic pathways may also contribute to withdrawal-induced attentional deficits, providing novel targets to alleviate the cognitive symptoms of withdrawal during quit attempts. PMID:28243714

Improving Blood Monitoring of Enzymes as Biomarkers of Risk from Anticholinergic Pesticides and Chemical Warfare Agents

DTIC Science & Technology

2007-10-01

agent exposure --monitoring blood cholinesterase activity with the Test-mateTM OP Kit. In Technical Bulletin: Assay Techniques for Detection of Exposure ...at CHPPM and by the Ellman method at UC Davis. Second, DFP-treated whole blood was used to mimic OP exposure . RBC AChE activity was measured using... cholinesterase activity in mammalian blood and tissue. Bulletin of Environmental Contamination and Toxicology 77(6):785- 792 Arrieta DE, McCurdy

A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity.

PubMed

Bandhuvula, Padmavathi; Fyrst, Henrik; Saba, Julie D

2007-12-01

Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available omega(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.

A new assay system for guinea pig interferon biological activity.

PubMed

Yamamoto, Toshiko; Jeevan, Amminikutty; Ohishi, Kazue; Nojima, Yasuhiro; Umemori, Kiyoko; Yamamoto, Saburo; McMurray, David N

2002-07-01

We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

Effects of chlorpyrifos on the transcription of CYP3A cDNA, activity of acetylcholinesterase, and oxidative stress response of goldfish (Carassius auratus).

PubMed

Ma, Junguo; Liu, Yang; Niu, Daichun; Li, Xiaoyu

2015-04-01

Chlorpyrifos (CPF) is the widely used organophosphate pesticide in agriculture throughout the world. It has been found that CPF is relatively safe to human but highly toxic to fish. In this study, acute toxicity of CPF on goldfish was determined and then the transcription of goldfish cytochrome P450 (CYP) 3A was evaluated after 96 h of CPF exposure at concentrations of 15.3 [1/10 50% lethal concentration (LC50 )] or 51 μg L(-1) (1/3 LC50 ) of CPF. Meanwhile, the enzymatic activities of acetylcholinesterase (AChE), superoxide dismutase (SOD), and catalase (CAT), total antioxidant activity (T-AOC), and the contents of malondialdehyde (MDA) in the liver or brain of goldfish were also determined. The results of acute toxicity testing showed that the 96-h LC50 of CPF to the goldfish was 153 μg L(-1) . Moreover, a length sequence of 1243 bp CYP3A cDNA encoding for 413 amino acids from goldfish liver was cloned. Polymerase chain reaction results reveal that CPF exposure downregulates CYP 3A transcription in goldfish liver, suggesting that goldfish CYP 3A may be not involved in CPF bioactivation. Finally, the results of biochemical assays indicate that 96 h of CPF exposure remarkably inhibits AChE activity in fish liver or brain, alters hepatic antioxidant enzyme activities, decreases brain T-AOC, and causes lipid peroxidation in fish liver. These results suggest that oxidative stress might be involved in CPF toxicity on goldfish. Copyright © 2013 Wiley Periodicals, Inc.

Muscarinic Acetylcholine Receptor Localization and Activation Effects on Ganglion Response Properties

PubMed Central

Renna, Jordan M.; Amthor, Franklin R.; Keyser, Kent T.

2010-01-01

Purpose. The activation and blockade of muscarinic acetylcholine receptors (mAChRs) affects retinal ganglion cell light responses and firing rates. This study was undertaken to identify the full complement of mAChRs expressed in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties. Methods. RT-PCR, Western blot analysis, and immunohistochemistry were used to identify the complement and distribution of mAChRs in the rabbit retina. Extracellular electrophysiology was used to determine the effects of the activation or blockade of mAChRs on ganglion cell response properties. Results. RT-PCR of whole neural retina resulted in the amplification of mRNA transcripts for the m1 to m5 mAChR subtypes. Western blot and immunohistochemical analyses confirmed that all five mAChR subtypes were expressed by subpopulations of bipolar, amacrine, and ganglion cells in the rabbit retina, including subsets of cells in cholinergic and glycinergic circuits. Nonspecific muscarinic activation and blockade resulted in the class-specific modulation of maintained ganglion cell firing rates and light responses. Conclusions. The expression of mAChR subtypes on subsets of bipolar, amacrine, and ganglion cells provides a substrate for both enhancement and suppression of retinal responses via activation by cholinergic agents. Thus, the muscarinic cholinergic system in the retina may contribute to the modulation of complex stimuli. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes that are caused by decreased ACh in the retinas of Alzheimer's patients and the potential visual effects of anticholinergic treatments for ocular diseases. PMID:20042645

Draft Genome Sequence of Aldehyde-Degrading Strain Halomonas axialensis ACH-L-8

PubMed Central

Ye, Jun; Ren, Chong; Shan, Xiexie

2016-01-01

Halomonas axialensis ACH-L-8, a deep-sea strain isolated from the South China Sea, has the ability to degrade aldehydes. Here, we present an annotated draft genome sequence of this species, which could provide fundamental molecular information on the aldehydes-degrading mechanism. PMID:27081145

Development of radiohalogenated muscarinic ligands for the in vivo imaging of m-AChR by nuclear medicine techniques

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

McPherson, D.W.; Luo, H.; Knapp, F.F. Jr.

1994-06-01

Alterations in the density of acetylcholinergic muscarinic receptors (m-AChR) have been observed in various dementias. This has spurred interest in the development of radiohalogenated ligands which can be used for the non-invasive in vivo detection of m-AChR by nuclear medicine techniques. We have developed a new ligand 1-azabicyclo[2.2.2]oct-3-yl ({alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IQNP,12) which demonstrates high affinity for the muscarinic receptor. When labeled with radioiodine it has been shown to be selective and specific for m-ACHR. Initial studies on the separation and in vivo evaluation of the various isomers of IQNP have shown that the stereochemistry of the chiral centers and the configurationmore » around the double bond play an important role in m-AChR subtype specificity. In vivo evaluation of these stereoisomers demonstrate that E-(R,R)-IQNP has a high affinity for the M{sub 1} muscarinic subtype while Z-(R,R)-IQNP demonstrate a high affinity for M{sub 1} and M{sub 2} receptor subtypes. These data demonstrate IQNP (12) has potential for use in the non-evasive in vivo detection of m-AChR by single photon emission computed tomography (SPECT). A brominated analogue, ``BrQNP,`` in which the iodine has been replaced by a bromine atom, has also been prepared and was shown to block the in vivo uptake of IQNP in the brain and heart and therefore has potential for positron emission tomographic (PET) studies of m-AChR.« less

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

PubMed Central

Goel, Anisha; Santos, Filipe; de Vos, Willem M.; Teusink, Bas

2012-01-01

Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of Vmax over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis. PMID:22020503

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.

Inhibition of Cholinesterases and Some Pro-Oxidant induced Oxidative Stress in Rats Brain by Two Tomato (Lycopersicon Esculentum) Varieties

PubMed Central

Oboh, G.; Bakare, O.O.; Ademosun, A.O.; Akinyemi, A.J.; Olasehinde, T.A.

2015-01-01

This study sought to investigate the effects of two tomato varieties [Lycopersicon esculentum Mill. var. esculentum (ESC) and Lycopersicon esculentum Mill. var. cerasiforme (CER)] on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities in vitro. Phenolics content, carotenoids characterisation, inhibition of Fe2+ and quinolinic acid-induced malondialdehyde (MDA) production in rats brain homogenate and NO* scavenging abilities were also assesed in addition to the AChE and BChE inhibition assays. There was no significant difference in the AChE inhibitory ability of the samples, while CER had significantly higher BChE inhibitory activity. Furthermore, the tomatoes inhibited Fe2+ and quinolinic acid-induced MDA production and further exhibited antioxidant activities through their NO* scavenging abilities. There was no significant difference in the phenolic content of the samples, while significantly high amounts of lycopene were detected in the tomatoes. The cholinesterase-inhibition and antioxidant properties of the “tomatoes” could make them good dietary means for the management of neurodegenerative disorders.

Optimized UDP-glucuronosyltransferase (UGT) activity assay for trout liver S9 fractions

EPA Pesticide Factsheets

This publication provides an optimized UGT assay for trout liver S9 fractions which can be used to perform in vitro-in vivo extrapolations of measured UGT activityThis dataset is associated with the following publication:Ladd, M., P. Fitzsimmons , and J. Nichols. Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: Activity enhancement by alamethicin, a pore-forming peptide. XENOBIOTICA. Taylor & Francis, Inc., Philadelphia, PA, USA, 46(12): 1066-1075, (2016).

Influence of water temperature on acetylcholinesterase activity in the pacific tree frog (Hyla regilla)

USGS Publications Warehouse

Johnson, Catherine S.; Schwarzbach, Steven E.; Henderson, John D.; Wilson, Barry W.; Tjeerdema, Ronald S.

2005-01-01

This investigation evaluated whether acetylcholinesterase (AChE) in Pacific tree frogs (Hyla regilla) from different geographical locations was influenced by different temperatures during early aquatic life stages, independent of pesticide exposure. Tadpoles were collected from both a California coastal pond and a Sierra Nevada mountain range pond, USA. Groups of frogs from each location were raised in temperatures representative of either the Sierra Nevada (8°C) or the coastal (19°C) location. Metamorphs from both locations raised as tadpoles at 19°C had AChE activities of 42.3 and 38.7 nm/min/mg protein, while those raised as tadpoles at 8°C had activities of 26.9 and 28.2 nm/min/mg protein. A two-way analysis of variance revealed temperature to be the significant factor in determining AChE activity (F = 22.3, p < 0.001), although origin was not important (F = 0.09, p = 0.75). Interpretations regarding the influence of pesticides upon AChE activity in Pacific tree frogs must consider the influence of environmental temperature to enable cross-population comparisons.

Lead finding for acetyl cholinesterase inhibitors from natural origin: structure activity relationship and scope.

PubMed

Mukherjee, P K; Satheeshkumar, N; Venkatesh, P; Venkatesh, M

2011-03-01

Acetylcholinesterase (AChE) inhibitors are considered as promising therapeutic agents for the treatment of several neurological disorders such as Alzheimer's disease (AD), senile dementia, ataxia and myasthenia gravis. There are only few synthetic medicines with adverse effects, available for treatment of cognitive dysfunction and memory loss associated with these diseases. A variety of plants has been reported to possess AChE inhibitory activity and so may be relevant to the treatment of neurodegenerative disorders such as AD. Hence, developing potential AChE inhibitors from botanicals is the need of the day. This review will cover some of the promising acetylcholinesterase inhibitors isolated from plants with proven in vitro and in vivo activities with concern to their structure activity relationship.

Effect of methylmercury on acetylcholinestrase and serum cholinesterase activity in monkeys, Macaca fascicularis

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

Petruccioli, L.; Turillazzi, P.G.

1991-05-01

The consumption of fish and fish-derived products is the main pathway of human exposure to methylmercury (MeHg). Methylmercury levels vary widely in fish, depending on age, size, the position of the species in the food chain, and most of all, on pollution levels. MeHg affects the Acetylcholinesterase activity (AChE) and the serum Cholinesterase activity (BChE). Histoenzymatic studies showed that 100mg Methyoxyethylmercury chloride administered for 6 days to rats caused a reduction of AChE activity in the thalamus and an increase in different parts of the nervous central system. The present study aims at verifying whether the dose permitted by F.A.O.more » and doses 10 and 100 fold higher affect the Cholinesterase activity in primates, and whether there is a correlation between AChE and BChE.« less

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

PubMed

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

2015-11-01

Variations in the enzyme activity of acetylcholinesterase (AChE) in the presence of the nano-material, graphene oxide (GO), were investigated with the use of molecular spectroscopy UV-visible and fluorescence methods. From these studies, important kinetic parameters of the enzyme were extracted; these were the maximum reaction rate, Vm , and the Michaelis constant, Km . A comparison of these parameters indicated that GO inhibited the catalytic activity of the AChE because of the presence of the AChE-GO complex. The formation of this complex was confirmed with the use of fluorescence data, which was resolved with the use of the MCR-ALS chemometrics method. Furthermore, it was found that the resonance light-scattering (RLS) intensity of AChE changed in the presence of GO. On this basis, it was demonstrated that the relationship between AChE and GO was linear and such models were used for quantitative analyses of GO. Copyright © 2015 John Wiley & Sons, Ltd.

Evaluation of potential endocrine activity of 2,4-dichlorophenoxyacetic acid using in vitro assays.

PubMed

Coady, Katherine K; Kan, H Lynn; Schisler, Melissa R; Gollapudi, B Bhaskar; Neal, Barbara; Williams, Amy; LeBaron, Matthew J

2014-08-01

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated in five in vitro screening assays to assess the potential for interaction with the androgen, estrogen and steroidogenesis pathways in the endocrine system. The assays were conducted to meet the requirements of the in vitro component of Tier 1 of the United States Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), and included assays for estrogen receptor (ER) binding (rat uterine cytosol ER binding assay), ER-mediated transcriptional activation (HeLa-9903-ERα transactivation assay), androgen receptor (AR) binding (rat prostate cytosol AR binding assay), aromatase enzymatic activity inhibition (recombinant human CYP19 aromatase inhibition assay), and interference with steroidogenesis (H295R steroidogenesis assay). Results from these five assays demonstrated that 2,4-D does not have the potential to interact in vitro with the estrogen, androgen, or steroidogenesis pathways. These in vitro data are consistent with a corresponding lack of endocrine effects observed in apical in vivo animal studies, and thus provide important supporting data valuable in a comprehensive weight of evidence evaluation indicating a low potential of 2,4-D to interact with the endocrine system. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Rational design, synthesis and biological screening of triazine-triazolopyrimidine hybrids as multitarget anti-Alzheimer agents.

PubMed

Jameel, Ehtesham; Meena, Poonam; Maqbool, Mudasir; Kumar, Jitendra; Ahmed, Waqar; Mumtazuddin, Syed; Tiwari, Manisha; Hoda, Nasimul; Jayaram, B

2017-08-18

In our endeavor towards the development of potent multitarget ligands for the treatment of Alzheimer's disease, a series of triazine-triazolopyrimidine hybrids were designed, synthesized and characterized by various spectral techniques. Docking and scoring techniques were used to design the inhibitors and to display their interaction with key residues of active site. Organic synthesis relied upon convergent synthetic routes were mono and di-substituted triazines were connected with triazolopyrimidine using piperazine as a linker. In total, seventeen compounds were synthesized in which the di-substituted triazine-triazolopyrimidine derivatives 9a-d showed better acetylcholinesterase (AChE) inhibitory activity than the corresponding tri-substituted triazine-triazolopyrimidine derivatives 10a-f. Out of the disubstituted triazine-triazolopyrimidine based compounds, 9a and 9b showed encouraging inhibitory activity on AChE with IC 50 values 0.065 and 0.092 μM, respectively. Interestingly, 9a and 9b also demonstrated good inhibition selectivity towards AChE over BuChE by ∼28 folds. Furthermore, kinetic analysis and molecular modeling studies showed that 9a and 9b target both catalytic active site as well as peripheral anionic site of AChE. In addition, these derivatives effectively modulated Aβ self-aggregation as investigated through CD spectroscopy, ThT fluorescence assay and electron microscopy. Besides, these compounds exhibited potential antioxidants (2.15 and 2.91 trolox equivalent by ORAC assay) and metal chelating properties. In silico ADMET profiling highlighted that, these novel triazine derivatives have appropriate drug like properties and possess very low toxic effects in the primarily pharmacokinetic study. Overall, the multitarget profile exerted by these novel triazine molecules qualified them as potential anti-Alzheimer drug candidates in AD therapy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

PubMed

Tiefenbacher, S; Bohra, R; Amiral, J; Bowyer, A; Kitchen, S; Lochu, A; Rosén, S; Ezban, M

2017-10-01

Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA ® -Cephascreen ® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective

Vagus nerve stimulation mediates protection from kidney ischemia-reperfusion injury through α7nAChR+ splenocytes.

PubMed

Inoue, Tsuyoshi; Abe, Chikara; Sung, Sun-Sang J; Moscalu, Stefan; Jankowski, Jakub; Huang, Liping; Ye, Hong; Rosin, Diane L; Guyenet, Patrice G; Okusa, Mark D

2016-05-02

The nervous and immune systems interact in complex ways to maintain homeostasis and respond to stress or injury, and rapid nerve conduction can provide instantaneous input for modulating inflammation. The inflammatory reflex referred to as the cholinergic antiinflammatory pathway regulates innate and adaptive immunity, and modulation of this reflex by vagus nerve stimulation (VNS) is effective in various inflammatory disease models, such as rheumatoid arthritis and inflammatory bowel disease. Effectiveness of VNS in these models necessitates the integration of neural signals and α7 nicotinic acetylcholine receptors (α7nAChRs) on splenic macrophages. Here, we sought to determine whether electrical stimulation of the vagus nerve attenuates kidney ischemia-reperfusion injury (IRI), which promotes the release of proinflammatory molecules. Stimulation of vagal afferents or efferents in mice 24 hours before IRI markedly attenuated acute kidney injury (AKI) and decreased plasma TNF. Furthermore, this protection was abolished in animals in which splenectomy was performed 7 days before VNS and IRI. In mice lacking α7nAChR, prior VNS did not prevent IRI. Conversely, adoptive transfer of VNS-conditioned α7nAChR splenocytes conferred protection to recipient mice subjected to IRI. Together, these results demonstrate that VNS-mediated attenuation of AKI and systemic inflammation depends on α7nAChR-positive splenocytes.

Roles of uptake, biotransformation, and target site sensitivity in determining the differential toxicity of chlorpyrifos to second to fourth instar Chironomous riparius (Meigen)

USGS Publications Warehouse

Buchwalter, D.B.; Sandahl, J.F.; Jenkins, J.J.; Curtis, L.R.

2004-01-01

Early life stages of aquatic organisms tend to be more sensitive to various chemical contaminants than later life stages. This research attempted to identify the key biological factors that determined sensitivity differences among life stages of the aquatic insect Chironomous riparius. Specifically, second to fourth instar larvae were exposed in vivo to both low and high waterborne concentrations of chlorpyrifos to examine differences in accumulation rates, chlorpyrifos biotransformation, and overall sensitivity among instars. In vitro acetylcholinesterase (AChE) assays were performed with chlorpyrifos and the metabolite, chlorpyrifos-oxon, to investigate potential target site sensitivity differences among instars. Earlier instars accumulated chlorpyrifos more rapidly than later instars. There were no major differences among instars in the biotransformation rates of chlorpyrifos to the more polar metabolites, chlorpyrifos-oxon, and chlorpyridinol (TCP). Homogenate AChE activities from second to fourth instar larvae were refractory to chlorpyrifos, even at high concentrations. In contrast, homogenate AChE activities were responsive in a dose-dependent manner to chlorpyrifos-oxon. In general, it appeared that chlorpyrifos sensitivity differences among second to fourth instar C. riparius were largely determined by differences in uptake rates. In terms of AChE depression, fourth instar homogenates were more sensitive to chlorpyrifos and chlorpyrifos-oxon than earlier instars. However, basal AChE activity in fourth instar larvae was significantly higher than basal AChE activity in second to third instar larvae, which could potentially offset the apparent increased sensitivity to the oxon. ?? 2003 Elsevier B.V. All rights reserved.

Juncaceae species as sources of innovative bioactive compounds for the food industry: In vitro antioxidant activity, neuroprotective properties and in silico studies.

PubMed

Rodrigues, Maria João; Gangadhar, Katkam N; Zengin, Gokhan; Mollica, Adriano; Varela, João; Barreira, Luísa; Custódio, Luísa

2017-09-01

Several Juncus species are traditionally used as sedative and to treat health problems like insomnia. This work was based on the hypothesis that Juncus acutus, J. maritimus and J. inflexus may have molecules with bioactivities relevant for the improvement of cognitive functions and thus with potential use as food additives and/or nutraceuticals. Therefore leaves and roots extracts of those species were evaluated for radical scavenging (RSA) and metal chelating activities, and for in vitro inhibition of acetyl-(AChE) and butyrylcholinesterase (BuChE). The bioactive compound was isolated and identified by HPLC-DAD, and its anticholinesterase capacity was determined by different assays. Docking studies were performed to elucidate its inhibitory mechanism. The dichloromethane root extract of J. acutus had the highest RSA against DPPH and ABTS radicals, and the dichloromethane extract of J. maritimus leaves had the uppermost FRAP. The dichloromethane extract from J. acutus leaves had the strongest BuChE inhibition. Juncunol was the bioactive compound, exhibiting dual anticholinesterase capacity on enzyme-based assays and AChE inhibition in neuronal and glial cells in vitro. Molecular docking studies indicate juncunol as a competitive reversible inhibitor. Our results suggest that Juncus spp. can be sources of bioactive compounds with application in the food industry as cognitive-enhancer nutraceuticals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Curcumin administration suppress acetylcholinesterase gene expression in cadmium treated rats.

PubMed

Akinyemi, Ayodele Jacob; Oboh, Ganiyu; Fadaka, Adewale Oluwaseun; Olatunji, Babawale Peter; Akomolafe, Seun

2017-09-01

Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes have been reported to exert anticholinesterase potential with limited information on how they regulate acetylcholinesterase (AChE) gene expression. Hence, this study sought to evaluate the effect of curcumin on cerebral cortex acetylcholinesterase (AChE) activity and their mRNA gene expression level in cadmium (Cd)-treated rats. Furthermore, in vitro effect of different concentrations of curcumin (1-5μg/mL) on rat cerebral cortex AChE activity was assessed. Animals were divided into six groups (n=6): group 1 serve as control (without Cd) and receive saline/vehicle, group 2 receive saline plus curcumin at 25mg/kg, group 3 receive saline plus curcumin 50mg/kg, group 4 receive Cd plus vehicle, group 5 receive Cd plus curcumin at 25mg/kg and group 6 receive Cd plus curcumin at 50mg/kg. Rats received Cd (2.5mg/kg) and curcumin (25 and 50mg/kg, respectively) by oral gavage for 7days. Acetylcholinesterase activity was measured by Ellman's method and AChE expression was carried out by a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assay. We observed that acute administration of Cd increased acetylcholinesterase activity and in addition caused a significant (P<0.05) increase in AChE mRNA levels in whole cerebral cortex when compared to control group. However, co-treatment with curcumin inhibited AChE activity and alters AChE mRNA levels when compared to Cd-treated group. In addition, curcumin inhibits rat cerebral cortex AChE activity in vitro. In conclusion, curcumin exhibit anti-acetylcholinesterase activity and suppressed AChE mRNA gene expression level in Cd exposed rats, thus providing some biochemical and molecular evidence on the therapeutic effect of this turmeric-derived compound in treating neurological disorders including Alzheimer's disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Install active/passive neutron examination and assay (APNEA)

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

Not Available

1996-04-01

This document describes activities pertinent to the installation of the prototype Active/Passive Neutron Examination and Assay (APNEA) system built in Area 336 into its specially designed trailer. It also documents the basic theory of operation, design and protective features, basic personnel training, and the proposed characterization site location at Lockheed Martin Specialty Components, Inc., (Specialty Components) with the estimated 10 mrem/year boundary. Additionally, the document includes the Preventive Change Analysis (PCA) form, and a checklist of items for verification prior to unrestricted system use.

Attenuated nicotine-like effects of varenicline but not other nicotinic ACh receptor agonists in monkeys receiving nicotine daily.

PubMed

Cunningham, Colin S; Moerke, Megan J; Javors, Martin A; Carroll, F Ivy; McMahon, Lance R

2016-12-01

Chronic treatment can differentially impact the effects of pharmacologically related drugs that differ in receptor selectivity and efficacy. The impact of daily nicotine treatment on the effects of nicotinic ACh receptor (nAChR) agonists was examined in two groups of rhesus monkeys discriminating nicotine (1.78 mg·kg -1 base weight) from saline. One group received additional nicotine treatment post-session (1.78 mg·kg -1 administered five times daily, each dose 2 h apart; i.e. Daily group), and the second group did not (Intermittent group). Daily repeated nicotine treatment produced a time-related increase in saliva cotinine. There was no significant difference in the ED 50 values of the nicotine discriminative stimulus between the Daily and Intermittent group. Mecamylamine antagonized the effects of nicotine, whereas dihydro-β-erythroidine did not. Midazolam produced 0% nicotine-lever responding. The nAChR agonists epibatidine, RTI-36, cytisine and varenicline produced >96% nicotine-lever responding in the Intermittent group. The respective maximum effects in the Daily group were 100, 72, 59 and 28%, which shows that the ability of varenicline to produce nicotine-like responding was selectively decreased in the Daily as compared with the Intermittent group. When combined with nicotine, both varenicline and cytisine increased the potency of nicotine to produce discriminative stimulus effects. Nicotine treatment has a greater impact on the sensitivity to the effects of varenicline as compared with some other nAChR agonists. Collectively, these results strongly suggest that varenicline differs from nicotine in its selectivity for multiple nAChR subtypes. © 2016 The British Pharmacological Society.

Attenuated nicotine‐like effects of varenicline but not other nicotinic ACh receptor agonists in monkeys receiving nicotine daily

PubMed Central

Cunningham, Colin S; Moerke, Megan J; Javors, Martin A; Carroll, F Ivy

2016-01-01

Background and Purpose Chronic treatment can differentially impact the effects of pharmacologically related drugs that differ in receptor selectivity and efficacy. Experimental Approach The impact of daily nicotine treatment on the effects of nicotinic ACh receptor (nAChR) agonists was examined in two groups of rhesus monkeys discriminating nicotine (1.78 mg·kg−1 base weight) from saline. One group received additional nicotine treatment post‐session (1.78 mg·kg−1 administered five times daily, each dose 2 h apart; i.e. Daily group), and the second group did not (Intermittent group). Key Results Daily repeated nicotine treatment produced a time‐related increase in saliva cotinine. There was no significant difference in the ED50 values of the nicotine discriminative stimulus between the Daily and Intermittent group. Mecamylamine antagonized the effects of nicotine, whereas dihydro‐β‐erythroidine did not. Midazolam produced 0% nicotine‐lever responding. The nAChR agonists epibatidine, RTI‐36, cytisine and varenicline produced >96% nicotine‐lever responding in the Intermittent group. The respective maximum effects in the Daily group were 100, 72, 59 and 28%, which shows that the ability of varenicline to produce nicotine‐like responding was selectively decreased in the Daily as compared with the Intermittent group. When combined with nicotine, both varenicline and cytisine increased the potency of nicotine to produce discriminative stimulus effects. Conclusion and Implications Nicotine treatment has a greater impact on the sensitivity to the effects of varenicline as compared with some other nAChR agonists. Collectively, these results strongly suggest that varenicline differs from nicotine in its selectivity for multiple nAChR subtypes. PMID:27667659

nAChR dysfunction as a common substrate for schizophrenia and comorbid nicotine addiction: current trends and perspectives

PubMed Central

Parikh, Vinay; Kutlu, Munir Gunes; Gould, Thomas J.

2016-01-01

Introduction The prevalence of tobacco use in the population with schizophrenia is enormously high. Moreover, nicotine dependence is found to be associated with symptom severity and poor outcome in patients with schizophrenia. The neurobiological mechanisms that explain schizophrenia-nicotine dependence comorbidity are not known. This study systematically reviews the evidence highlighting the contribution of nicotinic acetylcholine receptors (nAChRs) to nicotine abuse in schizophrenia. Methods Electronic data bases (Medline, Google Scholar, and Web of Science) were searched using the selected key words that match the aims set forth for this review. A total of 275 articles were used for the qualitative synthesis of this review. Results Substantial evidence from preclinical and clinical studies indicated that dysregulation of α7 and β2-subunit containing nAChRs account for the cognitive and affective symptoms of schizophrenia and nicotine use may represent a strategy to remediate these symptoms. Additionally, recent meta-analyses proposed that early tobacco use may itself increase the risk of developing schizophrenia. Genetic studies demonstrating that nAChR dysfunction that may act as a shared vulnerability factor for comorbid tobacco dependence and schizophrenia were found to support this view. The development of nAChR modulators was considered an effective therapeutic strategy to ameliorate psychiatric symptoms and to promote smoking cessation in schizophrenia patients. Conclusions The relationship between schizophrenia and smoking is complex. While the debate for the self-medication versus addiction vulnerability hypothesis continues, it is widely accepted that a dysfunction in the central nAChRs represent a common substrate for various symptoms of schizophrenia and comorbid nicotine dependence. PMID:26803692

Development of a spontaneously active dorsal root ganglia assay using multiwell multielectrode arrays

PubMed Central

Newberry, Kim; Wang, Shuya; Hoque, Nina; Kiss, Laszlo; Ahlijanian, Michael K.; Herrington, James

2016-01-01

In vitro phenotypic assays of sensory neuron activity are important tools for identifying potential analgesic compounds. These assays are typically characterized by hyperexcitable and/or abnormally, spontaneously active cells. Whereas manual electrophysiology experiments provide high-resolution biophysical data to characterize both in vitro models and potential therapeutic modalities (e.g., action potential characteristics, the role of specific ion channels, and receptors), these techniques are hampered by their low throughput. We have established a spontaneously active dorsal root ganglia (DRG) platform using multiwell multielectrode arrays (MEAs) that greatly increase the ability to evaluate the effects of multiple compounds and conditions on DRG excitability within the context of a cellular network. We show that spontaneous DRG firing can be attenuated with selective Na+ and Ca2+ channel blockers, as well as enhanced with K+ channel blockers. In addition, spontaneous activity can be augmented with both the transient receptor potential cation channel subfamily V member 1 agonist capsaicin and the peptide bradykinin and completely blocked with neurokinin receptor antagonists. Finally, we validated the use of this assay by demonstrating that commonly used neuropathic pain therapeutics suppress DRG spontaneous activity. Overall, we have optimized primary rat DRG cells on a multiwell MEA platform to generate and characterize spontaneously active cultures that have the potential to be used as an in vitro phenotypic assay to evaluate potential therapeutics in rodent models of pain. PMID:27052585

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

NASA Astrophysics Data System (ADS)

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

1988-06-01

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

77 FR 40148 - Proposed Collection of Information: ACH Vendor/Miscellaneous Payment Enrollment Form

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-06

.../Miscellaneous Payment Enrollment Form AGENCY: Financial Management Service, Fiscal Service, Treasury. ACTION: Notice and request for comments. SUMMARY: The Financial Management Service, as part of its continuing... Financial Management Service solicits comments concerning the SF 3881 ``ACH Vendor/Miscellaneous Payment...

Parkinson's disease (PD) with dementia and falls is improved by AChEI? A preliminary study report.

PubMed

Lauretani, Fulvio; Galuppo, Laura; Costantino, Cosimo; Ticinesi, Andrea; Ceda, Gianpaolo; Ruffini, Livio; Nardelli, Anna; Maggio, Marcello

2016-06-01

Advanced PD is often associated with cognitive impairment and frequent falls. We describe a suggestive case report of PD associated with mild cognitive impairment (MCI) and falls. The aim of our study was to test alteration in balance potentially related to use of acetylcholinesterase inhibitor (AchEI). We address this hypothesis after keeping the patient in stable dosage of dopamine agonist. We describe an initial pharmacological management in a 78-year-old man affected by Parkinson disease (PD) associated with mild cognitive impairment (MCI) and history of falls. The diagnosis of PD was also confirmed by SPECT with DATSCAN, after CT-brain exclusion of potential other causes of the symptoms. Cognitive and motor performances of the patient were initially evaluated by Mini Mental Examination State Examination (MMSE), Short Physical Performance Battery (SPPB) and Romberg test. We also recorded gait parameters using an accelerometer, while balance and stability were assessed by stabilometric platform with open and closed eyes. We repeated cognitive and motor tests and gait and balance evaluation after stable dosage of dopamine agonist before and after introduction of AchEI. After starting dopamine agonist therapy, there was a significant improvement in gait parameters (speed, stride/min, stride length, swing duration, and decrease in gait cycle duration and rolling duration). When stable dosage of dopamine agonist was reached, AchEI was introduced obtaining not only a significant improvement of cognitive performance, but also a significant positive change in balance. We hypothesize that AchEI could improve stability, balance and postural instability in addition to cognitive performance in PD with MCI and balance deficits.

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

PubMed

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

2016-05-01

Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

The role of the RhoA/ROCK pathway in gender-dependent differences in gastric smooth muscle contraction.

PubMed

Al-Shboul, Othman

2016-01-01

Gender-related differences in various gastric functions and diseases have been reported, with women having a higher prevalence of gastrointestinal disturbances than men. The aim of this study was to investigate sex-dependent differences in activation of the Rho-associated protein kinase (ROCK; RhoA/Rho kinase) pathway and muscle contraction in the stomach using single gastric smooth muscle cells (GSMC) from male and female Sprague-Dawley rats. Expression of ROCK1 and ROCK2 protein and acetylcholine (ACh)-induced activation of RhoA and ROCK were measured using a specifically designed enzyme-linked immunosorbent assay and activity assay kits, respectively. Contraction of a single GSMC was measured by scanning micrometry in the presence or absence of the ROCK inhibitor Y27632 dihydrochloride. ACh-induced activation of RhoA and ROCK and subsequent contraction were greater in male rats than in female rats but neither was related to differences in the expression of ROCK1 or ROCK2 or total RhoA amount. Most important, Y27632 inhibited and abolished differences in ACh-induced contraction in both sexes. In conclusion, increased ACh-induced contraction in the GSMC of male rats is attributable to greater RhoA/ROCK activation independent of differences in the expression of ROCK isoforms or total RhoA.

The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function.

PubMed

Araud, Tanguy; Graw, Sharon; Berger, Ralph; Lee, Michael; Neveu, Estele; Bertrand, Daniel; Leonard, Sherry

2011-10-15

The human α7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is a candidate gene for schizophrenia and an important drug target for cognitive deficits in the disorder. Activation of the α7*nAChR, results in opening of the channel and entry of mono- and divalent cations, including Ca(2+), that presynaptically participates to neurotransmitter release and postsynaptically to down-stream changes in gene expression. Schizophrenic patients have low levels of α7*nAChR, as measured by binding of the ligand [(125)I]-α-bungarotoxin (I-BTX). The structure of the gene, CHRNA7, is complex. During evolution, CHRNA7 was partially duplicated as a chimeric gene (CHRFAM7A), which is expressed in the human brain and elsewhere in the body. The association between a 2bp deletion in CHRFAM7A and schizophrenia suggested that this duplicate gene might contribute to cognitive impairment. To examine the putative contribution of CHRFAM7A on receptor function, co-expression of α7 and the duplicate genes was carried out in cell lines and Xenopus oocytes. Expression of the duplicate alone yielded protein expression but no functional receptor and co-expression with α7 caused a significant reduction of the amplitude of the ACh-evoked currents. Reduced current amplitude was not correlated with a reduction of I-BTX binding, suggesting the presence of non-functional (ACh-silent) receptors. This hypothesis is supported by a larger increase of the ACh-evoked current by the allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596) in cells expressing the duplicate than in the control. These results suggest that CHRFAM7A acts as a dominant negative modulator of CHRNA7 function and is critical for receptor regulation in humans. Copyright © 2011 Elsevier Inc. All rights reserved.

The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function

PubMed Central

Araud, Tanguy; Graw, Sharon; Berger, Ralph; Lee, Michael; Neveu, Estelle; Bertrand, Daniel; Leonard, Sherry

2011-01-01

The human α7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is a candidate gene for schizophrenia and an important drug target for cognitive deficits in the disorder. Activation of the α7*nAChR, results in opening of the channel and entry of mono- and divalent cations, including Ca++, that presynaptically participates to neurotransmitter release and postsynaptically to down-stream changes in gene expression. Schizophrenic patients have low levels of α7*nAChR, as measured by binding of the ligand [125I]-α-bungarotoxin (I-BTX). The structure of the gene, CHRNA7, is complex. During evolution, CHRNA7 was partially duplicated as a chimeric gene (CHRFAM7A), which is expressed in the human brain and elsewhere in the body. The association between a 2bp deletion in CHRFAM7A and schizophrenia suggested that this duplicate gene might contribute to cognitive impairment. To examine the putative contribution of CHRFAM7A on receptor function, co-expression of α7 and the duplicate genes was carried out in cell lines and Xenopus oocytes. Expression of the duplicate alone yielded protein expression but no functional receptor and co-expression with α7 caused a significant reduction of the amplitude of the ACh-evoked currents. Reduced current amplitude was not correlated with a reduction of I-BTX binding, suggesting the presence of non-functional (ACh-silent) receptors. This hypothesis is supported by a larger increase of the ACh-evoked current by the allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596) in cells expressing the duplicate than in the control. These results suggest that CHRFAM7A acts as a dominant negative modulator of CHRNA7 function and is critical for receptor regulation in humans. PMID:21718690

Automated chromatographic laccase-mediator-system activity assay.

PubMed

Anders, Nico; Schelden, Maximilian; Roth, Simon; Spiess, Antje C

2017-08-01

To study the interaction of laccases, mediators, and substrates in laccase-mediator systems (LMS), an on-line measurement was developed using high performance anion exchange chromatography equipped with a CarboPac™ PA 100 column coupled to pulsed amperometric detection (HPAEC-PAD). The developed method was optimized for overall chromatographic run time (45 to 120 min) and automated sample drawing. As an example, the Trametes versicolor laccase induced oxidation of 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (adlerol) using 1-hydroxybenzotriazole (HBT) as mediator was measured and analyzed on-line. Since the Au electrode of the PAD detects only hydroxyl group containing substances with a limit of detection being in the milligram/liter range, not all products are measureable. Therefore, this method was applied for the quantification of adlerol, and-based on adlerol conversion-for the quantification of the LMS activity at a specific T. versicolor laccase/HBT ratio. The automated chromatographic activity assay allowed for a defined reaction start of all laccase-mediator-system reactions mixtures, and the LMS reaction progress was automatically monitored for 48 h. The automatization enabled an integrated monitoring overnight and over-weekend and minimized all manual errors such as pipetting of solutions accordingly. The activity of the LMS based on adlerol consumption was determined to 0.47 U/mg protein for a laccase/mediator ratio of 1.75 U laccase/g HBT. In the future, the automated method will allow for a fast screening of combinations of laccases, mediators, and substrates which are efficient for lignin modification. In particular, it allows for a fast and easy quantification of the oxidizing activity of an LMS on a lignin-related substrate which is not covered by typical colorimetric laccase assays. ᅟ.

Enzymatic assay for calmodulins based on plant NAD kinase activity

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

Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required formore » 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.« less

Acetylcholinesterase inhibitory activity of Thai traditional nootropic remedy and its herbal ingredients.

PubMed

Tappayuthpijarn, Pimolvan; Itharat, Arunporn; Makchuchit, Sunita

2011-12-01

The incidence of Alzheimer disease (AD) is increasing every year in accordance with the increasing of elderly population and could pose significant health problems in the future. The use of medicinal plants as an alternative prevention or even for a possible treatment of the AD is, therefore, becoming an interesting research issue. Acetylcholinesterase (AChE) inhibitors are well-known drugs commonly used in the treatment of AD. The aim of the present study was to screen for AChE inhibitory activity of the Thai traditional nootropic recipe and its herbal ingredients. The results showed that ethanolic extracts of four out of twenty-five herbs i.e. Stephania pierrei Diels. Kaempfera parviflora Wall. ex Baker, Stephania venosa (Blume) Spreng, Piper nigrum L at 0.1 mg/mL showed % AChE inhibition of 89, 64, 59, 50; the IC50 were 6, 21, 29, 30 microg/mL respectively. The other herbs as well as combination of the whole recipe had no synergistic inhibitory effect on AChE activity. However some plants revealed antioxidant activity. More research should have be performed on this local wisdom remedy to verify the uses in scientific term.

Evidence for aging theories from the study of a hunter-gatherer people (Ache of Paraguay).

PubMed

Libertini, G

2013-09-01

In the late seventies, a small tribal population of Paraguay, the Ache, living under natural conditions, was studied. Data from this population turn out to be useful for considerations about evolutionary hypotheses on the aging phenomenon. 1) Ache show an age-related increasing mortality, which strongly limits the mean duration of life, as observed in other studies on mammal and bird species. 2) According to current theories on aging, in the wild very few or no individual reach old age and, so, aging cannot be directly influenced by natural selection. However, data from our population show that a significant proportion of the population reaches in the wild 60 and 70 years of age. 3) Data from Ache are also in agreement with the observation about an inverse correlation between extrinsic mortality and deaths due to the age-related increasing mortality. 4) For many gerontologists, the age-related decline of vital functions is a consequence of the gradual decline of cell turnover, genetically determined and regulated by the declining duplication capacities of stem cells. The current interpretation is that these restrictions are a general defense against the proliferation of any tumoral mass. However, among wild Ache cancer is virtually unknown in non-elderly subjects, and only among older individuals are there deaths attributable to oncological diseases. Moreover, fitness decline begins long before oncological diseases have fatal effects in significant numbers. This completely disproves the current hypothesis, because a supposed defense against a deadly disease cannot exterminate a population before the disease begins to kill. These data are consistent with similar data from other species studied under natural conditions, and they bring new arguments against the non-adaptive interpretation of aging and in support of the adaptive interpretation.

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities.

PubMed

Hitomi, Kiyotaka; Kitamura, Miyako; Alea, Mileidys Perez; Ceylan, Ismail; Thomas, Vincent; El Alaoui, Saïd

2009-11-15

Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.

Cholinesterase inhibitors from Cleistocalyx operculatus buds.

PubMed

Min, Byung Sun; Cuong, To Dao; Lee, Joo-Sang; Shin, Beom-Soo; Woo, Mi Hee; Hung, Tran Manh

2010-10-01

Five flavonoids, myricetin-3'-methylether 3-O-β-D: -galactopyranoside (1), myricetin-3',5'-dimethylether 3-O-β-D: -galactopyranoside (2), quercetin (3), kaempferol (4), and tamarixetin (5) were isolated from the buds of Cleistocalyx operculatus (Myrtaceae). The chemical structures of these compounds were determined on the basis of spectroscopic analyses, including 2D NMR. Their anti-Alzheimer effects were evaluated via acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity assays. All five compounds 1-5 showed potential inhibitory activities against AChE with IC(50) values of 19.9, 37.8, 25.9, 30.4 and 22.3 μM, respectively, while compounds 1, 3, 4 and 5 also possessed BChE inhibitory activity with IC(50) values of 152.5, 177.8, 62.5, and 160.6 μM, respectively.

Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology.

PubMed

Niessen, K V; Muschik, S; Langguth, F; Rappenglück, S; Seeger, T; Thiermann, H; Worek, F

2016-04-15

Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100 μM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

New multipotent tetracyclic tacrines with neuroprotective activity.

PubMed

Marco-Contelles, José; León, Rafael; de los Ríos, Cristóbal; García, Antonio G; López, Manuela G; Villarroya, Mercedes

2006-12-15

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues (5-13) are described. Compounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca(2+) overload or free radical induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor (11) is 50-fold less potent than tacrine], but proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement experiments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential agents that can prevent the aggregation of beta-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient neuroprotective agent against 70mM K(+) and 60microM H(2)O(2). Based on these results, some of these molecules can be considered as lead candidates for the further development of anti-Alzheimer drugs.

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

Molecular docking and ex vivo and in vitro anticholinesterase activity studies of Salvia sp. and highlighted rosmarinic acid.

PubMed

Demirezer, Lütfiye Ömür; Gürbüz, Perihan; Kelicen Uğur, Emine Pelin; Bodur, Mine; Özenver, Nadire; Uz, Ayse; Güvenalp, Zühal

2015-01-01

To evaluate acetylcholinesterase (AChE) inhibitory activity and antioxidant capacity of the major molecule from Salvia sp., rosmarinic acid, as a drug candidate molecule for treatment of Alzheimer disease (AD). The AChE inhibitory activity of different extracts from Salvia trichoclada, Salvia verticillata, and Salvia fruticosa was determined by the Ellman and isolated guinea pig ileum methods, and the antioxidant capacity was determined with DPPH. The AChE inhibitory activity of the major molecule rosmarinic acid was determined by in silico docking and isolated guinea pig ileum methods. The methanol extract of Salvia trichoclada showed the highest inhibition on AChE. The same extract and rosmarinic acid showed significant contraction responses on isolated guinea pig ileum. All the extracts and rosmarinic acid showed high radical scavenging capacities. Docking results of rosmarinic acid showed high affinity to the selected target, AChE. In this study in vitro and ex vivo studies and in silico docking research of rosmarinic acid were used simultaneously for the first time. Rosmarinic acid showed promising results in all the methods tested.

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

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

Yang Dongren; Howard, Angela; Bruun, Donald

2008-04-01

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