In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio)

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides. © 2012 Elsevier Ltd. All rights reserved.

A useful panel for the diagnosis of Hirschsprung disease in rectal biopsies: calretinin immunostaining and acetylcholinesterase histochesmistry

The pathological evaluation of rectal biopsies for the diagnosis of Hirschsprung disease has been a challenging issue. We analyzed prospectively the usefulness of calretinin immunostaining and acetylcholinesterase (AChE) histochesmistry in rectal biopsies for the diagnosis of Hirschsprung disease. Frozen tissue samples from 43 patients were used for AChE histochemistry and paraffin-embedded sections for calretinin immunohistochemistry and conventional histology (hematoxylin and eosin [H&E]). Activity for AChE, was demonstrated in 13 of 43 cases, and the absence of immunoreactivity for calretinin was observed in 14 of 43 cases. Conventional histology (H&E) did not reveal ganglion cells in 24 of 43 cases. The results on calretinin were in good agreement with AChE according to the κ index (0.946; P < .001) and presented significantly higher specificity (96.7 × 63.3; P < .002) and accuracy (97.6 × 74.4; P < .003) when compared with conventional histology (H&E). The final diagnosis of Hirschsprung disease was confirmed in 13 of 43 patients who were submitted to surgical treatment. The results of the present study indicate that calretinin can be a good tool in ruling out the diagnosis of Hirschsprung disease, by showing positive staining in ganglion cells and intrinsic nerve fibers, whereas AChE is useful in confirming the diagnosis of Hirschsprung disease, by revealing activity of this enzyme in hypertrophied nerve fibers. The association between calretinin and AChE can be a useful panel for the histopathologic evaluation of rectal biopsies for the diagnosis of Hirschsprung disease. © 2013 Elsevier Inc. All rights reserved.

Design, synthesis and characterization of a hexapeptide bio-inspired by acetylcholinesterase and its interaction with pesticide dichlorvos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chemical Composition of the Bark of Tetrapterys mucronata and Identification of Acetylcholinesterase Inhibitory Constituents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antifungals and acetylcholinesterase inhibitors from the stem bark of Croton heliotropiifolius

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antioxidant, anti-acetylcholinesterase and cytotoxic activities of ethanol extracts of peel, pulp and seeds of exotic Brazilian fruits Antioxidant, anti-acetylcholinesterase and cytotoxic activities in fruits

Ethanol extracts of powdered genipap (Genipa americana L), umbu (Spondia tuberosa A.) and siriguela (Spondia purpurea L) prepared from separate pulp, seeds and peel were investigated for their (i) antioxidant capacity, which was evaluated by various known methods; (ii) acetylcholinesterase (AChE) inhibitory activity; and (iii) cytotoxic effect on corneal epithelial cells of sheep. The highest values of total phenolic content were obtained with peel and seed extracts. Siriguela and umbu (seeds and peel) extracts displayed the highest antioxidant activities. Lipid peroxidation assays using mimetic biomembranes and mouse liver homogenates indicated that genipap pulp is a promising antioxidant. The investigation of phenols and organic acid contents revealed the presence of quercetin, citric and quinic acids, chlorogenic acid derivatives, among others, in several extracts, with the highest amount found in siriguela seeds. Genipap pulp and siriguela seed ethanol extracts presented an AChE inhibition zone similar to that of the positive control, carbachol. AChE inhibition assay with chlorogenic acid, one of the main constituents of siriguela seeds, revealed that this acid showed activity similar to that of the control physostigmine. These data suggest that these extracts are potentially important antioxidant supplements for the everyday human diet, pharmaceutical and cosmetic industries. (C) 2012 Elsevier Ltd. All rights reserved.

Pyridine Alkaloids from Senna multijuga As Acetylcholinesterase Inhibitors

As part of an ongoing research project on Senna and Cassia species, five new pyridine alkaloids, namely, 12'-hydroxy-7'-multijuguinol (1), 12'-hydroxy-8'-multijuguinol (2), methyl multijuguinate (3), 7'-multijuguinol (4), and 8'-multijuguinol (5), were isolated from the leaves of Senna multijuga (syn. Cassia multijuga). Their structures were elucidated on the basis of spectroscopic data analysis. Mass spectrometry was used for confirmation of the positions of the hydroxy groups in the side-chains of 1, 2, 4, and 5. All compounds exhibited weak in vitro acetylcholinesterase inhibitory activity as compared with the standard compound physostigmine.

Electrochemical detection of carbamate pesticides in fruit and vegetables with a biosensor based on acetylcholinesterase immobilised on a composite of polyaniline-carbon nanotubes

A sensitive electrochemical acetylcholinesterase (AChE) biosensor was successfully developed on polyaniline (PANI) and multi-walled carbon nanotubes (MWCNTs) core-shell modified glassy carbon electrode (GC), and used to detect carbamate pesticides in fruit and vegetables (apple, broccoli and cabbage). The pesticide biosensors were applied in the detection of carbaryl and methomyl pesticides in food samples using chronoamperometry (CA). The GC/MWCNT/PANI/AChE biosensor exhibited detection limits of 1.4 and 0.95 mu mol L-1, respectively, for carbaryl and methomyl. These detection limits were below the allowable concentrations set by Brazilian regulation standards for the samples in which these pesticides were analysed. Reproducibility and repeatability values of 2.6% and 3.2%, respectively, were obtained in the conventional procedure. The proposed biosensor was successfully applied in the determination of carbamate pesticides in cabbage, broccoli and apple samples without any spiking procedure. The obtained results were in full agreement with those from the HPLC procedure. (C) 2012 Elsevier Ltd. All rights reserved.

Motion-insensitive determination of B1+ amplitudes based on the bloch-siegert shift in single voxels of moving organs including the human heart.

PURPOSE To reliably determine the amplitude of the transmit radiofrequency ( B1+) field in moving organs like the liver and heart, where most current techniques are usually not feasible. METHODS B1+ field measurement based on the Bloch-Siegert shift induced by a pair of Fermi pulses in a double-triggered modified Point RESolved Spectroscopy (PRESS) sequence with motion-compensated crusher gradients has been developed. Performance of the sequence was tested in moving phantoms and in muscle, liver, and heart of six healthy volunteers each, using different arrangements of transmit/receive coils. RESULTS B1+ determination in a moving phantom was almost independent of type and amplitude of the motion and agreed well with theory. In vivo, repeated measurements led to very small coefficients of variance (CV) if the amplitude of the Fermi pulse was chosen above an appropriate level (CV in muscle 0.6%, liver 1.6%, heart 2.3% with moderate amplitude of the Fermi pulses and 1.2% with stronger Fermi pulses). CONCLUSION The proposed sequence shows a very robust determination of B1+ in a single voxel even under challenging conditions (transmission with a surface coil or measurements in the heart without breath-hold). Magn Reson Med, 2015. © 2015 Wiley Periodicals, Inc.

MECHANISMS OF SPECIES DIFFERENCES IN BRAIN ACETYLCHOLINESTERASE SENSITIVITY TO ORGANOPHOSPHATE INHIBITION

In vitro incubation of acetylcholinesterase from brain tissue of several species with organophosphate compounds indicated that the concentrations required to inhibit 50% of acetylcholinesterase activity (IC(,50)) differed from species to species for the same compound (Murphy, et al., 1968; Andersen, et al., 1972, 1977 and 1978).^ The hypothesis that non-specific binding proteins (Lauwerys and Murphy, 1969a,b) exerts a protective effect on acetylcholinesterase, and thus cause the differences observed in IC(,50) studies was tested by a ('3)H-DFP binding experiment. It was found that differences in the amount of non-specific binding protein cannot explain the observed differences observed in IC(,50) studies.^ An alternative hypothesis, that acetylcholinesterase from different species have different affinities for binding and/or different rates of phosphorylation by organophosphate insecticides was tested by determining the apparent affinity constant (k(,a)) and apparent rate of phosphorylation (k(,p)). Kinetic studies indicated that acetylcholinesterases from different species have different sensitivities to inhibition by organophosphate insecticides, and the differences are due to different affinities for binding and/or different rates of phosphorylation by the same organophosphate compound.^ Studies of the spontaneous reactivation of acetylcholinesterase after inhibition by organophosphate insecticides also indicated that acetylcholinesterases from different species have different rates and extents of spontaneous reactivation. This further substantiates the hypothesis that acetylcholinesterases from different species have different kinetic characteristics with respect to organophosphate insecticides inhibition.^ Eleven paraoxon analogs were synthesized for a quantitative structure-activity relationship study. It was found that the electron-withdrawing power ((sigma)) and hydrophobicity ((PARAGR)) of the substituent are important in determining the anti-cholinesterase activity of paraoxon analogs. Thus, predictions of species differences in acetylcholinesterase sensitivities to paraoxon analogs can be made if the physicochemical parameters ((sigma) and (PARAGR)) of the substituents are known.^ In another approach, i.e. enzyme modeling, the sensitivity of rat brain acetylcholinesterase to organophosphate insecticides was used as the independent variable to predict the sensitivities of acetylcholinesterases from other species to the same compound. Regression equations were derived for each species based on nineteen organophosphate insecticides studied. It was found, that in addition to paraoxon analogs, this method is also applicable to other organophosphate compounds with wide variations in structure. Thus, the sensitivities of acetylcholinesterases from other species can also be predicted from the sensitivity of rat brain acetylcholinesterase. ^

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by α-toxin

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho → Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal α-toxin, including an α-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [γ-32P]ATP to α-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.