Abstract Acetylcholinesterase Activity Research Articles

Effect of very Low Frequency Electromagnetic Radiation on Acetylcholine Esterase Activity

Acetylcholinesterase (AChE) is the enzyme that controls the acetylcholine (ACh) concentrations in cholinergic synaptic clefts by hydrolyzing ACh to choline and acetate. In this paper, we investigate the effect of very low frequency (VLF) electromagnetic field (EMF) radiation on AChE activity. AChE was exposed to VLF-EMFs with electric field intensity ranged from 0 to 200V/m and exposure time ranged from 0 to 120min. The results demonstrated that the VLF-EMF increased enzyme activity when the intensity ranged from 80V/m to 100V/m as well as exposure time ranged from 60min to 80min. And the AChE activity decreased when the intensity ranged from 120V/m to 140V/m and exposure time ranged from 20min to 120min. Thus, those results are significant in clinical treatments.

To Measure the Absorbance at 328nm by Ultraviolet-Visible Spectrophotometer - an Alternative Pathway to Measure the Activity of Acetylcholinesterase

Acetylcholinesterase (AChE) is an important enzyme in mammalian nervous systems. The Ellman assay is usually used to measure AChE activity and calculate the inhibition rate by measure the absorbance at 412nm by UV-Vis spectrophotometer. However, an alternative absorbance at 329nm which assigned as S-S of 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) was found, and it negatively correlated with the absorbance at 412nm. Therefore, the absorbance at 329nm was also possible used to measure AChE activity and calculate the inhibition rate. In addition, the reaction time should be considered in the process to transfer the relationship of these two absorbance values, because Cu (II) would combine with 5'-mercapto-2'-nitrobenzoic acid (5-MNBA).

Characterization of acetylcholinesterase in Hong Kong oyster (Crassostrea hongkongensis) from South China Sea

Acetylcholinesterase (AChE) activity has been used to evaluate the exposure of mollusk bivalves to organophosphates, carbamate pesticides, and heavy metals. Crassostrea hongkongensis is a Hong Kong endemic oyster, and has a high commercial value along the coastal area of South China. The use of this species as a bio-indicator of the marine environment, and the use of AChE activity measurements in tissues of C. hongkongensis require prior characterization of AChE in this species. Here, we report that gill tissue contains the highest AChE activity in C. hongkongensis, and that the molecular form of AChE is most likely to be a dimeric form. In addition, the effect of the pesticide acephate on AChE activity in the gill of C. hongkongensis was analyzed, and the mean inhibition concentration (IC50) value was determined. This study suggests that AChE activity in the gill tissue of C. hongkongensis might be used as a biomarker in monitoring organophosphate contamination in the marine fauna of South China.

Treatment with endotracheal therapeutics after sarin microinstillation inhalation exposure increases blood cholinesterase levels in guinea pigs

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the blood and tissues of animals that are treated with a number of endotracheally aerosolized therapeutics for protection against inhalation toxicity to sarin. Therapeutics included, aerosolized atropine methyl bromide (AMB), scopolamine or combination of AMB with salbutamol, sphingosine 1-phosphate, keratinocyte growth factor, adenosine A1 receptor antisense oligonucleotide (EPI2010), 2,3-diacetyloxybenzoic acid (2,3 DABA), oxycyte, and survanta. Guinea pigs exposed to 677.4 mg/m3 or 846.5 mg/m3 (1.2 LCt50) sarin for 4 min using a microinstillation inhalation exposure technique and treated 1 min later with the aerosolized therapeutics. Treatment with all therapeutics significantly increased the survival rate with no convulsions throughout the 24 h study period. Blood AChE activity determined using acetylthiocholine as substrate showed 20% activity remaining in sarin-exposed animals compare to controls. In aerosolized AMB and scopolamine-treated animals the remaining AChE activity was significantly higher (45–60%) compared to sarin-exposed animals (p < 0.05). Similarly, treatment with all the combination therapeutics resulted in significant increase in blood AChE activity in comparison to sarin-exposed animals although the increases varied between treatments (p < 0.05). BChE activity was increased after treatment with aerosolized therapeutics but was lesser in magnitude compared to AChE activity changes. Various tissues showed elevated AChE activity after therapeutic treatment of sarin-exposed animals. Increased AChE and BChE activities in animals treated with nasal therapeutics suggest that enhanced breathing and reduced respiratory toxicity/lung injury possibly contribute to rapid normalization of chemical warfare nerve agent inhibited cholinesterases.

Alteration of acetylcholinesterase activity in Semele solida (MOLLUSCA: SEMELIDAE) as a biochemical response to coastal anthropogenic impact

Acetylcholinesterase (AChE) activity was analyzed as a molecular marker indicative of exposure to organophosphorus pesticide residues in individuals of the endemic clam species Semele solida in selected coastal locations of Chile's VIII Region. AChE activity was assayed in clams from (i) Penco Beach near the mouth of the Andalién River (Concepción Bay), (ii) Lenga Beach, near the mouth of the Lenga Estuary (San Vicente Bay), and (iii) Coliumo Beach, near the mouth of the Coliumo Estuary (Coliumo Bay). We also analyzed variations in protein content of clam hemolymph, and variability in the activity of AChE in relation to the sizes of the individual clams sampled. Collection of the clams was done using routine methods, during the spring of 2005, the period during which the use of pesticides is typically intensified in the surrounding forestry and agriculture. The results showed no significant correlation of AChE activity with either the size of the clam, or with the concentration of proteins in the hemolymph. The lowest AChE activity was observed in clams from mouth of the Andalién River (187.5 ± 34.9 Umin−1) which was significantly less than that measured in specimens collected near the mouth of the Coliumo Estuary and the mouth of the Lenga Estuary. A very close relation was observed between the degree of anthropogenic inputs, ocean dynamics, and alterations in AChE activity in S. solida. This clam appears to be a useful indicator species, and AChE activity a sensitive marker for the presence of xenobiotics.

Structural Characterization of Acetylcholinesterase 1 from the Sand Fly &lt;I&gt;Lutzomyia longipalpis&lt;/I&gt; (Diptera: Psychodidae)

Acetylcholinesterase (AChE) plays a key role in cholinergic impulse transmission, and it is the target enzyme for organophosphorus and carbamate insecticides. Two genes, AceI and AceII, have been characterized from different insect species, and point mutations in either gene can lead to significant resistance to these classes of insecticides. In this report, we describe the partial characterization of the AceI gene from Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae), and we show that the possibility exists for the development of a resistant phenotype to organophosphates and carbamates in sand flies. Our results point to the presence of a single AceI gene in L. longipalpis (LlAce1) and that AChE activity is inhibited by organophosphorus at a concentration of 5 x 10(-5) M. Regarding insecticide resistance, analysis of the truncated LlAce1 cDNA suggests that a single missense mutation leading to a glycine-to-serine substitution at amino acid position 119 (G119S) may arise in L. longipalpis, similar to what has been detected in Anopheles gambiae s.s. Another missense mutation involved in resistant phenotypes, F331W, detected in Culex tritaeniorhynchus Giles, is less likely to occur in L. longipalpis, because it faces codon constraint in this sand fly species. Comparison of the three-dimensional structures of the deduced amino acid sequence of the truncated LLAChE1 with that of An. gambiae and Cx. tritaeniorhynchus also suggests that similar structural modifications due to the missense amino acid changes in the active site gorge are detected in all three insects.

Gene Transfer of Acetylcholinesterase Protects the Knockout Mouse From the Toxicity of DFP

Acetylcholinesterase (AChE) has a clear role in nerve impulse transmission. Organophosphorus esters are highly toxic chemicals used as pesticides, fire retardants, plasticizers, and chemical warfare agents. The acute toxicity of organophosphorus poisons is attributed to inhibition of AChE in nerve synapses. This leads to seizures, respiratory arrest, and death. Our goal was to find a new therapeutic for protection against the toxicity of organophosphates (OPs). We investigated the feasibility of using a gene therapy vector to deliver AChE over long time periods and in quantities sufficiently high to provide protection against diisopropylfluorophosphate (DFP) toxicity. We used the AChE-/- mouse for these studies because this mouse has no endogenous AChE activity (Xie et al., 2000). Any AChE activity found in tissues could only come from the viral vector.

Perlecan Helps Keep AChE in Place

Acetylcholinesterase (AChE) is expressed by innervated muscles at the site of motor neuron contact and is required for the termination of acetylcholine-mediated neurotransmission. A particular form of AChE that has a collagen tail is preferentially located at the neuromuscular junction (NMJ), but the mechanism by which it is localized to the NMJ is not well understood. Now, Arikawa-Hirasawa et al. have found that a complex extracellular heparan sulfate proteoglycan termed perlecan is required for proper localization of AChE to the synaptic basal lamina of the NMJ. Whereas muscle development, muscle innervation, and NMJ synapse formation were normal in perlecan-deficient mice, very little AChE was detected in the NMJ of the mutant mice. This result was confirmed in fluorescence assays where no AChE was observed in the NMJ of perlecan-deficient mice. However, the absence of perlecan in these mice did not affect their production of AChE mRNA and protein, because no difference in total AChE activity was detected in muscle homogenates from wild-type and perlecan-deficient mice. Nonetheless, perlecan-deficient mice exhibited poor locomotion and respiratory failure. Thus, perlecan appears to be required for the proper extracellular localization of AChE to the NMJ. E. Arikawa-Hirasawa, S. G. Rossi, R. L. Rotundo, Y. Yamada, Absence of acetylcholinesterase at the neuromuscular junctions of perlecan-null mice. Nature Neurosci. 5 , 119-123 (2002). [Online Journal]

Some blood biochemistry parameters during the cholinergic treatment of Alzheimer's disease.

Acetylcholinesterase (ACE) activity and lipid peroxidation (LPO) parameters were measured in the blood of patients with Alzheimer's disease (AD) during treatment with amiridine and gliatiline. Treatment was accompanied by inhibition of ACE. There was a statistically significant relationship between clinical efficacy and changes in ACE activity. AD was charactefized by significant changes in LPO parameters, with a three-fold increase in the level of primary oxidation products on the background of a sharp (seven-fold) increase in total lipid desaturatedness. There was a statistically significant relationship between ACE activity and the levels of primary oxidation products in the RBC of patients with AD before and after treatment with amiridine and gliatiline.

Relative Activity of Various Esterases in Six Pakistani Strains of the Lesser Grain Borer, Rhyzopertha dominica (Fabricius)

Acetylcholinesterase (AChE), arylesterase (AE), carboxylesterase (CE), and cholinesterase (ChE) activities were determined in six adult populations of the lesser grain borer, Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrichidae) collected from grain warehouses in various areas of Punjab, namely Lahore (L), Sialkot (S), Wazirabad (W), Multan (M), Chichawatni (C), and Karachi (K). The multiple forms of esterases were resolved by polyacrylamide gel electrophoresis (PAGE) and activities determined biochemically, using a spectrophotometer. All the strains had the same AChE activity except Sialkot strain, which had 46% more activity than the susceptible K strain. Similarly K, S, W, L, and C strains exhibited almost the same AE level, whereas the M strain showed 29% lower activity than K strain. The CE activity of L (352%), S (198%) and C (214%) strains and ChE activity of L (178%) and C (317%) strains were significantly higher than that of K strain. The total esterases of L, S and C strains showed respectively 152%, 63% and 73% higher activity than the susceptible K strain. The PAGE pattern of various esterases coincided with the biochemical analysis. The variable thickness of the bands in the gel indicated relative esterase induction that could be correlated with the development of resistance in R. dominica.

Metrifonate

Acetylcholinesterase (AChE) inhibitors are currently the most promising drugs available for the treatment of Alzheimer's disease (AD). Their efficacy is based on the cholinergic hypothesis of AD which links reduced levels of cerebral acetylcholine (ACh) with declining cognitive function in affected individuals. AChE inhibitors are believed to work by increasing the level of ACh in the synaptic cleft by binding to local AChE and preventing the hydrolysis of ACh. Metrifonate differs from other AChE inhibitors in that it is a prodrug which is, non-enzymatically converted in vivo to the active moiety 2,2-dichlorovinyl dimethylphosphate (DDVP). DDVP administered alone has a very short plasma elimination half-life, but small amounts released from metrifonate are sufficient to inhibit AChE activity in vivo. DDVP is an irreversible inhibitor of AChE and activity is maintained for several weeks. Metrifonate has been used as an anthelmintic since 1962 and has been under investigation as a treatment for the symptoms of AD since 1990. Randomised, placebo-controlled clinical trials, using a variety of dose regimens, have demonstrated that metrifonate produces a significant, but modest, improvement in the 3 domains of AD: cognition, behaviour and function. However, it should be noted that with some assessment instruments in some trials, the improvement' was actually a reduction in the rate of worsening of symptoms compared with placebo. Weekly and daily schedules, with and without loading doses, have been evaluated and the research supports a fixed daily dose of 40 to 50mg, administered to supply approximately 0.65 mg/kg. In studies of up to 6 months' duration, metrifonate was well tolerated, and adverse events were mild and predominantly gastrointestinal. In clinical practice it is likely that metrifonate will be administered for several years and long term monitoring of adverse events will be important to further define the drug's tolerability profile. Muscle weakness occurred in a dose-related fashion in 20 of 3000 patients taking part in long term trials, and in some patients respiratory support was required. The mechanism of muscle weakness is not well defined and requires further investigation. Conclusions: Clinical data support the use of metrifonate in patients with AD. However, the improvements noted are modest and to date there is no evidence that metrifonate is more effective than other currently approved AChE inhibitors. The unique pharmacokinetic/pharmacodynamic profile of metrifonate may endow the agent with some advantages over other therapies, but this has yet to be evaluated in comparative trials. Moreover, long term studies evaluating the effects of metrifonate on maintenance of independence are required. While the clinical future of metrifonate is uncertain, it is one of a small group of drugs that have been shown to improve the outlook of patients with AD. Metrifonate is administered orally and after absorption is nonenzymatically converted to the active compound 2,2-dichlorovinyl dimethylphosphate (DDVP), at neutral or alkaline pH. After a short reversible phase, DDVP irreversibly binds to acetylcholinesterase (AChE). With the exception of nicotinic receptors, metrifonate does not bind to neurotransmitter receptors or binding sites associated with ion channels. Therefore, the pharmacological action of metrifonate is through inhibition of AChE and a consequent increase in levels of acetylcholine (ACh).

Effect of aging on the activities of acetylcholinesterase, Na+,K(+)-ATPase and Mg(2+)-ATPase in rat pituitary and hypothalamus.

Acetylcholinesterase (AChE), Na+,K(+)-ATPase and Mg(2+)-ATPase activities were estimated in homogenised rat pituitary and hypothalamus of 4- and 22-month-old rats. AChE activity was not altered in the pituitary of aged compared to adult rats, while it was found decreased by about 40% in the hypothalamus. Na+,K(+)-ATPase activity remained stable in the hypothalamus, while it was decreased by about 38% in the pituitary. Mg(2+)-ATPase activity remained unchanged in the hypothalamus, but was increased by about 83% in the pituitary. This pituitary Na+,K(+)-ATPase inactivation may result in pathological mood and decreased neural excitability and metabolic energy production in aged animals. The age-related alterations of AChE, Na+,K(+)-ATPase and Mg(2+)-ATPase activities may reflect changes in secretion and responses of some hormones of pituitary and hypothalamus.

Asymmetric distribution of acetylcholinesterase in gravistimulated maize seedlings.

Acetylcholinesterase (AChE) activity has previously been studied by this laboratory and shown to occur at the interface between the stele and cortex of the mesocotyl of maize (Zea mays L.) seedlings. In this work we studied the distribution of AChE activity in 5-d-old maize seedlings following a gravity stimulus. After the stimulus, we found an asymmetric distribution of the enzyme in the coleoptile, the coleoptile node, and the mesocotyl of the stimulated seedlings using both histochemical and colorimetric methods for measuring the hydrolysis of acetylthiocholine. The hydrolytic capability of the esterase was greater on the lower side of the horizontally placed seedlings. Using the histochemical method, we localized the hydrolytic capability in the cortical cells around the vascular stele of the tissues. The hydrolytic activity was inhibited 80 to 90% by neostigmine, an inhibitor of AChE. When neostigmine was applied to the corn kernel, the gravity response of the seedling was inhibited and no enzyme-positive spots appeared in the gravity-stimulated seedlings. We believe these results indicate a role for AChE in the gravity response of maize seedlings.

Acetylcholinesterase from desert cobra (Walterinnesia aegyptia) venom: optimization and kinetics study.

Acetylcholinesterase (AChE) was investigated in Walterinnesia aegyptia venom and characterized with respect to its kinetic properties. It was found that 4.0 micrograms of crude venom protein and an incubation time of 4.0 min were suitable conditions for linearity of AChE activity at 25 degrees C. The optimum strength of the sodium phosphate buffer was 0.05 M, and the optimum pH was 7.75. The optimum temperature was 30 degrees C. The activation energy and the heat of activation were observed to be 6510 and 5922 cal/mole. The AChE was specific for acetylthiocholine but it did not hydrolyse butyrylthiocholine. The optimum substrate concentration was 3.0 mM but at higher substrate concentrations, the AChE activity declined. The ASCh concentration ranges for different orders of the reactions were determined and kinetic parameters (Km, Vmax, Kcat, and Ksp) were established at each order of the reaction.

Purification and biochemical characterization of acetylcholinesterase (AChE) from the excretory/secretory products ofTrichostrongylus colubriformis

Acetylcholinesterase (AChE) has been purified from the excretory/secretory (ES) products of Trichostrongylus colubriformis (using edrophonium chloride linked to epoxy-activated Sepharose) with yields of 40-50%. Purity was confirmed by polyacrylamide gel electrophoresis (using silver [protein] and Karnovsky [activity] stains) and measurement of specific AChE activity. Further analysis of the purified fractions by gel filtration and sucrose density gradient techniques revealed the existence of 2 forms of hydrophilic AChE (M(r) 189 and 80 kDa). From the data we deduce these to be the globular monomer and dimer, G1 and G2 forms of AChE. Inhibition studies using BW284C51, iso-OMPA and excess substrate, along with substrate specificity studies, show both forms to be true acetylcholinesterases. We are currently assessing the protective immunogenicity of purified AChE in sheep.

Acetylcholinesterase from the Horn Fly (Diptera: Muscidae): Distribution and Purification

Acetylcholinesterase (AChE) in newly emerged adults of the horn fly, Haematobia irritans (L.), was distributed mostly in the head (72.9%), less in the thorax (24.4%), and least in the abdomen (2.7%). The major portion of total AChE was membrane-bound and associated primarily with the microsomes. The enzyme was solubilized with phosphate buffer containing 0.5% (vol/vol) of Triton X-100 and purified with affinity chromatography. The extent of purification was about 217-fold for the membranous fraction and 329-fold for the soluble fraction of whole fly homogenates with recoveries of 55% and 79%, respectively. The extent of purification reached 600- and 784-fold for the membranous and the soluble fractions, respectively, in the collections of eluents with peaked activity of AChE. Electrophoresis revealed four bands of AChE activity in the membrane-bound preparation and three bands in the soluble preparation. Silver staining of the electrophoresed gels showed that the major proteins in purified AChE solutions corresponded with AChE bands.

The molecular forms of acetylcholinesterase in cerebrospinal fluid of normal subjects ? effect of aging

Acetylcholinesterase (AChE) activity is increased in human cerebrospinal fluid (CSF) during aging. The present study investigated whether the relative amounts of different molecular forms of CSF-AChE are also affected during aging. Thus, the CSF samples of healthy human subjects (age range 20-79 years, n = 23) were analyzed for sedimentation forms of AChE activity. Five different forms of AChe activity were detected in human CSF. The relative amount of tetrameric and dimeric globular forms, which are the main forms of AChE in CSF, were not related with age. Furthermore, the relative amount of monomeric globular and asymmetric forms which are minor forms of AChE in CSF did not seem to be related to age. Since total CSF-AChE activity is increased during aging, it seems to be due to the increased amounts of the tetrameric and dimeric forms of enzyme activity.

Necator americanus secretory acetylcholinesterase and its purification from excretory‐secretory products by affinity chromatography

Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthiocholine iodide (ATC), and its activity was inhibited by eserine (1.1 x 10(-8) M). Ancylostoma ceylanicum, another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophageal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenates and earlier developmental stages. On IEF, purified AChE resolved predominantly with a pl of 3.55; proteins with a similar pl were recognized by rabbit anti-AChE. IgG preparations of this antiserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host-parasite relationship.

Decrease in the rat bronchial acetylcholinesterase activity after toluene diisocyanate inhalation

Acetylcholinesterase (AChE) activity was measured in bronchial tissue homogenate and blood from rats subjected to single and repeated 4-h daily inhalation exposure to toluene diisocyanate (TDI) or control atmospheres. A single 4-h exposure to TDI in the concentration range of 0.7-4.3 ppm did not decrease AChE activity in bronchial tissue but a 4-h exposure to 0.6 ppm TDI, or greater, for two consecutive days did reduce this activity (19% to 33% of the controls). Increasing the level of exposure to TDI for two consecutive days from 0.6 to 4.0 ppm and extending the length of exposure to 1.2 ppm TDI from 2 to 4, 9 or 14 days produced no further decrease in bronchial ACHE activity. Throughout these experiments, blood AChE activity remained unchanged. In rats exposed to 0.3 or 1 ppm TDI for 3 weeks, staining of the bronchial smooth muscle for AChE was reduced (36% of the controls) after exposure to 1 ppm TDI. These results support, for the first time, in vivo and localized anti-AChE activity by TDI.

Cerebrospinal fluid gradients of acetylcholinesterase and butyrylcholinesterase activity in healthy aging

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in 13 sequential 2 ml aliquots of cerebrospinal fluid (CSF) obtained by lumbar puncture from 7 young and 7 elderly healthy normal subjects. The slopes of the rostrocaudal gradients of AChE and BChE were calculated and compared to those of total protein concentration and the major dopaminergic metabolite homovanillic acid (HVA), for which a pronounced rostrocaudal gradient (with highest concentrations of HVA in more rostral CSF) is consistent with HVA originating primarily from the brain. AChE activity was higher in more caudal fractions of young, but not elderly subjects and there was a significant difference between the mean AChE gradient slopes in the young and old groups. These results suggest that the spinal cord makes an important contribution to AChE activity in lumbar CSF. Furthermore, the absence of a negative AChE gradient in elderly subjects may be the result of a greater rate of entry of cerebral AChE into CSF, possibly as a consequence of an increased ventricular surface area and shorter diffusion distances in atrophic elderly brains. In contrast to AChE, BChE activity and total protein concentrations were higher in more caudal CSF fractions of not only young but also old subjects. In addition, there was a significant correlation between the gradient slopes of BChE activity and total protein concentrations, suggesting that the majority of BChE activity in lumbar CSF derives from the same source as the majority of total protein, namely plasma. The diffuse (i.e. brain and spinal cord) origin of AChE in lumbar CSF would explain the relatively modest changes in lumbar CSF AChE activity in diseases involving certain central cholinergic systems, most notably Alzheimer's disease.