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

PubMed

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

2013-11-01

Disulfated and trisulfated steroids have been synthesized from cholesterol and their acetylcholinesterase inhibitory activity has been evaluated. In our studies we have found that the activity was not only dependent on the location of the sulfate groups but on their configurations. 2β,3α,6α-trihydroxy-5α-cholestan-6-one trisulfate (18) was the most active steroid with an IC50 value of 15.48 μM comparable to that of 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (1). Both compounds were found to be less active than the reference compound eserine. The butyrylcholinesterase activity of 1 and 18 was one magnitude lower than that against acetylcholinesterase revealing a selective inhibitor profile. Copyright © 2013 Elsevier Inc. All rights reserved.

Cholinergic inhibition of adrenergic neurosecretion in the rabbit iris-ciliary body

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

Jumblatt, J.E.; North, G.T.

The prejunctional effects of cholinergic agents on release of norepinephrine from sympathetic nerve endings were investigated in the isolated, superfused rabbit iris-ciliary body. Stimulation-evoked release of /sup 3/H-norepinephrine was inhibited by the cholinergic agonists methacholine, oxotremorine, muscarine, carbamylcholine and acetylcholine (plus eserine), but was unmodified by pilocarpine or nicotine. Agonist-induced inhibition was antagonized selectively by atropine, indicating a muscarinic response. Atropine alone markedly enhanced norepinephrine release, revealing considerable tonic activation of prejunctional cholinergic receptors in this system. Prejunctional inhibition by carbamylcholine was found to completely override the facilitative action of forskolin or 8-bromo-cyclic AMP on neurotransmitter release. Cholinergic and alphamore » 2-adrenergic effects on neurosecretion were non-additive, suggesting that the underlying receptors coexist at neurotransmitter release sites.« less

Autonomic regulation of mucociliary transport rate in the oesophagus of the frog, Rana temporaria.

PubMed Central

Morley, J; Sanjar, S

1984-01-01

Transport of lead particles along the mucosal surface of the frog oesophagus has been measured by direct observation with the aid of video recording. Electrical stimulation of the vagus nerve increased the rate of particle transport. This acceleration was suppressed by atropine or by hexamethonium. Acetylcholine and other parasympathomimetic agents accelerated particle transport rate. Such acceleration was abolished by atropine. Nicotine increased the rate of particle transport and this effect was suppressed by hexamethonium or by atropine. Atropine did not significantly alter basal particle transport rate. Neither basal particle transport rate nor the response to vagal nerve stimulation were affected by eserine. Adrenaline, noradrenaline or isoprenaline did not affect basal particle transport rate. Adrenaline or noradrenaline were without effect on the increased particle transport rate due to electrical stimulation of the vagus. PMID:6332901

ACETYL CHOLINESTERASE AND BUTYRYL CHOLINESTERASE INhIBITORY ACTIVITIES OF ZALEYA PENTANDRA.

PubMed

Afzal, Samina; Chaudhry, Bashir Ahmad; Afzal, Khurram; Saeed, Javeria; Akash, Sajd Hamid; Qadir, Muhammad Imran

2017-05-01

The aim of this study was to reveal acetyl cholinesterase (AchE) and butyryl cholinesterase (BchE) inhibitory activities of Zaleya pentandra. The aerial parts of the plant were air, freeze-dried and powdered. The extraction was carried out with methanol at room temperature for 24 h. The extract was concentrated on rotavapor and fractioned by column chromatography. The isolation and purification afforded amorphous solid which was subjected to physical, chemical and spectroscopic techniques i.e., UV, IR, H-NMR, "C-NMR and HREI-MS for the structure elucidation of the isolated compound. The compound was concluded as "Pentandradione" a novel compound. AchE and BchE inhibitory activities were estimated. The result showed that the isolated extract possessed significant activity against butyryl cholinesterase as compared to standard eserine while the extract lacks acetyl cholinesterase inhibitory activity.

Diagnosing and treating Phthirus pubis palpebrarum.

PubMed

Couch, J M; Green, W R; Hirst, L W; de la Cruz, Z C

1982-01-01

Phthiriasis palpebrarum is an uncommon cause of blepharitis and conjunctivitis and may easily be overlooked. A high index of suspicion and careful examination of the patient's lid margins and eyelashes will lead to the proper diagnosis. Treatment is best accomplished by careful removal of the lice and nits (louse eggs) from the patient's lashes. Local application of a pediculocide such as yellow mercuric oxide N.F. 1% ophthalmic ointment applied twice daily for one week or 0.25% physostigmine (Eserine) ointment applied twice daily for a minimum of ten days, to the lid margins should be considered when the total removal of Phthirus pubis and nits is not possible mechanically. Body hair should be examined for infestation with lice and treated with gamma benzene hexachloride shampoo. This medication should be used with caution in infants, children and pregnant women. Family members, sexual contacts, and close companions should be examined and treated appropriately; clothing, linen and personal items should be disinfected with heat of 50 degrees C for 30 minutes.

[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.

Suitability of cholinesterase of polychaete Diopatra neapolitana as biomarker of exposure to pesticides: In vitro characterization.

PubMed

Mennillo, Elvira; Casu, Valentina; Tardelli, Federica; De Marchi, Lucia; Freitas, Rosa; Pretti, Carlo

2017-01-01

Cholinesterases of Diopatra neapolitana were characterized for their activity in whole body and different body segments (apical, intermediate, posterior), substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (K m and V max ) and in vitro response to model inhibitors (eserine hemisulfate, isoOMPA, BW284C51) and carbamates (carbofuran, methomyl, aldicarb and carbaryl). Results showed that the rate of hydrolysis for acetyl- and propionylthiocholine was higher in the posterior segment than the apical/intermediate segments and whole body. Cholinesterases of D. neapolitana showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was poorly hydrolyzed indicating, together with the absence of inhibition by the specific inhibitor and the absence of reactive bands in native electrophoresis, a lack of an active butyrylcholinesterase, differently than that observed in other Annelida species. The degree of inhibition by selected carbamates of cholinesterase activity with propionylthiocholine as substrate was higher than that observed with ATChI-ChE activity; aldicarb showed the highest inhibitory effect. Copyright © 2016 Elsevier Inc. All rights reserved.

Early differential cell death and survival mechanisms initiate and contribute to the development of OPIDN: A study of molecular, cellular, and anatomical parameters

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

Damodaran, T.V., E-mail: tdamodar@nccu.edu; Pharmacology and Cancer biology, Duke University Medical Center, Durham, NC; Dept of Biology, North Carolina Central University, Durham, NC 27707

Organophosphorus-ester induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral, distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken that results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 ml/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. Themore » hens were euthanized at different time points such as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum, brainstem and spinal cord were quickly dissected and frozen for mRNA (northern) studies. Northern blots were probed with BCL2, GADD45, beta actin, and 28S RNA to investigate their expression pattern. Another set of hens was treated for a series of time points and perfused with phosphate buffered saline and fixative for histological studies. Various staining protocols such as Hematoxylin and Eosin (H and E); Sevier-Munger; Cresyl echt Violet for Nissl substance; and Gallocynin stain for Nissl granules were used to assess various patterns of cell death and degenerative changes. Complex cell death mechanisms may be involved in the neuronal and axonal degeneration. These data indicate altered and differential mRNA expressions of BCL2 (anti apoptotic gene) and GADD45 (DNA damage inducible gene) in various tissues. Increased cell death and other degenerative changes noted in the susceptible regions (spinal cord and cerebellum) than the resistant region (cerebrum), may indicate complex molecular pathways via altered BCL2 and GADD45 gene expression, causing the homeostatic imbalance between cell survival and cell death mechanisms. Semi quantitative analysis revealed that the order of severity of damage declines from the spino-cerebellar, ventral, and dorsal tract respectively, suggesting neuroanatomical specificity. Thus, early activation of cell death and cell survival processes may play significant role in the clinical progression and syndromic clinical feature presentation of OPIDN. -- Highlights: Black-Right-Pointing-Pointer Multiple mechanisms of neurodegeneration were indicated in a study on OPIDN model. Black-Right-Pointing-Pointer Altered expressions of BCL2 and GADD45 were recorded in various tissues of CNS. Black-Right-Pointing-Pointer Multiple anomalous cellular (neuronal and astroglial) features were recorded. Black-Right-Pointing-Pointer Anatomical specificity of the neurodegeneration was described.« less

Acetylcholine, carbachol, and GABA induce no detectable change in the length of isolated outer hair cells.

PubMed

Bobbin, R P; Fallon, M; Puel, J L; Bryant, G; Bledsoe, S C; Zajic, G; Schacht, J

1990-08-01

The mechanical and electrical properties of cochlear outer hair cells (OHCs) are suggested to modulate transduction by inner hair cells. These properties of OHCs are presumably regulated by efferent neurons which use several transmitters including acetylcholine (Ach) and gamma aminobutyric acid (GABA). Since it had been suggested that Ach causes isolated OHCs to shorten visibly, this study was designed to investigate whether GABA also alters the length of OHCs. OHCs were isolated from the guinea pig cochlea by mechanical dispersion after collagenase treatment. Cells were initially selected by strict morphological criteria. In addition they were only included in further studies if they attained a constant length during 10 min of superfusion with buffer solution. Neither GABA (20 microM: 100 microM), Ach (5 mM; 10 microM with 10 microM eserine) or carbachol (10 microM; 100 microM) altered OHC length when applied in iso-osmotic Hank's balanced salt solution (total number of cells tested, 72). If a change in length occurred it must have been smaller than 0.3 microns, our detection ability. In contrast, high potassium and variations in osmolarity changed hair cell length by 3-10% in agreement with other reports.

Electrolyte and protein secretion by the perfused rabbit mandibular gland stimulated with acetylcholine or catecholamines

PubMed Central

Case, R. M.; Conigrave, A. D.; Novak, I.; Young, J. A.

1980-01-01

1. A method is described for the isolation and vascular perfusion in vitro of the mandibular gland of the rabbit. The perfusate is a physiological salt solution containing glucose as the only metabolic substrate. 2. During perfusion with solutions containing acetylcholine, the gland secretes vigorously at a rate and in a manner similar to that seen in vivo. Although the gland becomes oedematous during perfusion, the extent of this oedema appears to have no influence on secretory ability: the perfused glands were capable of functioning for at least 4 h, and often for more than 6 h. 3. Acetylcholine evoked a small secretory response at a concentration of 8 × 10-9 mol l-1 and a maximum response at 8 × 10-7 mol l-1. Eserine (2 × 10-5 mol l-1) evoked secretory responses comparable to those evoked by acetylcholine in a concentration of 8 × 10-9 mol l-1. Secretion, whether unstimulated or evoked by acetylcholine or eserine, could be blocked completely by atropine. 4. During prolonged stimulation with acetylcholine, the fluid secretory response declined rapidly over a period of about 15 min from an initial high value to a much lower plateau value. After 3 or more hours of stimulation, the secretory response began once more to decline, this time towards zero. If, before the second period of decline begins, stimulation is interrupted for about 30 min, the gland recovers its initial responsiveness to further stimulation with acetylcholine. 5. The Na, K, Cl and HCO3 concentrations and the osmolality of acetylcholine evoked saliva exhibited flow-dependency similar to that seen in vivo. The concentrations of Na and Cl, but not K and HCO3, increased by about 25 mmol l-1 during periods of prolonged stimulation with acetylcholine even though the salivary secretory rate was constant. The concentrations of K and HCO3, but not Na and Cl, increased progressively as the concentration of infused acetylcholine was increased. 6. Salivary protein secretion increased with increasing concentrations of acetylcholine to a greater extent than did fluid secretion. During continuous stimulation, the rate of protein secretion fell off much faster than the rate of fluid secretion. 7. The β-adrenergic agonist isoproterenol evoked a fluid secretory response only equal to about 5% of that evoked by acetylcholine, but still the response declined during continued stimulation. The electrolyte composition of isoproterenol-evoked saliva was vastly different from that evoked by acetylcholine, being particularly rich in K and HCO3. The isoproterenol-evoked saliva was also extremely rich in protein so that the total protein secretion evoked by isoproterenol was much greater than that evoked by acetylcholine. 8. The α-adrenergic agonist phenylephrine was without stimulatory effect on salivary fluid secretion and caused a reduction in the secretory response to acetylcholine. The drug had little or no effect on the electrolyte content of acetylcholine-evoked saliva and appeared to reduce its protein content. PMID:7381794

Interaction of Triton X-100 with acyl pocket of butyrylcholinesterase: effect on esterase activity and inhibitor sensitivity of the enzyme.

PubMed

Jaganathan, L; Boopathy, R

1998-06-01

The effect of non-ionic detergents like Triton X-100, Lubrol PX, Brij 35 and Tween 80 on the esterase activity and inhibitor sensitivity of human serum butyrylcholinesterase (BuChE) were studied. The results showed that though BuChE is not a detergent dependent enzyme, the esterase activity and inhibitor sensitivity of it can be modulated by the presence of detergents. All the detergents caused a marginal activation of the esterase activity. The presence of Lubrol PX, Brij 35 or Tween 80 did not affect the 50% molar inhibition concentration (IC50) of the inhibitors tested. But in the presence of Triton X-100 the IC50 values were increased for neostigmine, eserine and tetraisopropylpyrophosphoramide (acylation site interacting inhibitors), whereas for inhibitors like ethopropazine, imipramine and procainamide (choline binding pocket specific inhibitors) the IC50 values were unaltered. In addition, in the presence of Triton X-100 the bimolecular reaction constant for phosphorylation reaction (ki) of BuChE for the acyl pocket specific tetraisopropylpyrophosphoramide was reduced. Triton X-100 partially protected BuChE against this tetraisopropylpyrophosphoramide inactivation. These results indicate that Triton X-100 by interacting with the acyl pocket hydrophobic region is able to activate the esterase activity of BuChE. Further it reduces the capacity of the enzyme to react with inhibitors that inactivate it by interacting with the serine residue of the acylation site.

Anti-inflammatory and anticholinesterase activity of six flavonoids isolated from Polygonum and Dorstenia species.

PubMed

Dzoyem, Jean Paul; Nkuete, Antoine H L; Ngameni, Barthelemy; Eloff, Jacobus N

2017-10-01

This study was aimed at investigating the anti-inflammatory and anticholinesterase activity of six naturally occurring flavonoids: (-) pinostrobin (1), 2',4'-dihydroxy-3',6'-dimethoxychalcone (2), 6-8-diprenyleriodictyol (3), isobavachalcone (4), 4-hydroxylonchocarpin (5) and 6-prenylapigenin (6). These compounds were isolated from Dorstenia and Polygonum species used traditionally to treat pain. The anti-inflammatory activity was determined by using the Griess assay and the 15-lipoxygenase inhibitory activity was determined with the ferrous oxidation-xylenol orange assay. Acetylcholinesterase inhibition was determined by the Ellman's method. At the lowest concentration tested (3.12 µg/ml), compounds 2, 3 and 4 had significant NO inhibitory activity with 90.71, 84.65 and 79.57 % inhibition respectively compared to the positive control quercetin (67.93 %). At this concentration there was no significant cytotoxicity against macrophages with 91.67, 72.86 and 70.86 % cell viability respectively, compared to 73.1 % for quercetin. Compound 4 had the most potent lipoxygenase inhibitory activity (IC 50 of 25.92 µg/ml). With the exception of (-) pinostrobin (1), all the flavonoids had selective anticholinesterase activity with IC 50 values ranging between 5.93 and 8.76 µg/ml compared to the IC 50 4.94 µg/ml of eserine the positive control. These results indicate that the studied flavonoids especially isobavachalcone are potential anti-inflammatory natural products that may have the potential to be developed as therapeutic agents against inflammatory conditions and even Alzheimer's disease.

Plasma Cholinesterase Activity in Female Green Turtles Chelonia mydas Nesting in Laguna de Terminos, Mexico Related to Organochlorine Pesticides in Their Eggs.

PubMed

Rivas-Hernández, Gerardo; May-Uc, Yaneli; Noreña-Barroso, Elsa; Cobos-Gasca, Víctor; Rodríguez-Fuentes, Gabriela

2018-01-01

The inhibition of cholinesterase (ChE) activity has been used as a biomarker of exposure to organophosphate and carbamate insecticides. ChE of nesting female green turtles (Chelonia mydas) were biochemically characterized using two substrates, acetylthiocholine iodide and butyrylthiocholine iodide, and three ChE inhibitors (eserine sulfate, BW284C51 and iso-OMPA). The results indicated that BChE is the predominant plasma ChE in female C. mydas, but with atypical properties that differ from those found in human BChE. Eggs from green turtles nesting at two sites in Laguna de Terminos contained µg g -1 (wet weight) quantities of organochlorine (OC) pesticides. Drins (aldrin, dieldrin, endrin, endrin ketone, endrin aldehyde) were found at the highest concentrations with no significant differences in the concentrations in eggs collected at the two sampling sites. A negative relationship was found between levels of OC pesticides in eggs and BChE activity in the plasma of female turtles laying the eggs. Since OC pesticides are not cholinesterase inhibitors, we hypothesized that this inverse relationship may be related to an antagonistic effect between OCs and organophosphate pesticides and mobilization of OCs from the fatty tissues of the female turtles into their eggs. However, further study is required to verify the hypothesis. It is also possible that other contaminants, such as petroleum hydrocarbons are responsible for the modulation of cholinesterase activity in female turtles.

Bile salt-stimulated lipase: an animal model for human lactation

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

Hamosh, M.; Freed, L.M.; York, C.M.

1986-03-01

To date, bile salt-stimulated lipase (BSSL), an important digestive enzyme for the newborn, has only been described in the milk of primates - human and gorilla. The authors report the presence of BSSL in milks of dog and cat. Serial collections from two dogs (day 1-49) and cats (day 5-57) were analyzed for BSSL activity using a /sup 3/H-triolein emulsion as substrate. Comparable analyses of pooled, term human milk were made for comparison. BSSL activity in individual dog milks (x = 32.0; range: 4.8-107.4 U/ml) was similar, while that in cat milk (x = 6.6; range: 2.2-16.9 U/ml) was lowermore » than in human milk (x = 37.0; range: 10-80 U/ml; n = 35). Longitudinal patterns for BSSL differed depending upon the enzyme source. Dog, cat and human milk BSSL all showed a neutral to alkaline pH optimum (pH 7.0-8.4), stability at low pH, and 95-100% inhibition (at concentrations of 0.6 mM) by NaCl and eserine. BSSL activity from all sources had an obligate requirement for primary bile salts. These data are the first to show BSSL activity in milk from mammals other than human and gorilla. Presence of BSSL in nonprimate milk will permit the careful study of BSSL biology in the mammary gland as well as its role in neonatal fat digestion.« less

Swimming speed alteration of Artemia sp. and Brachionus plicatilis as a sub-lethal behavioural end-point for ecotoxicological surveys.

PubMed

Garaventa, Francesca; Gambardella, Chiara; Di Fino, Alessio; Pittore, Massimiliano; Faimali, Marco

2010-03-01

In this study, we investigated the possibility to improve a new behavioural bioassay (Swimming Speed Alteration test-SSA test) using larvae of marine cyst-forming organisms: e.g. the brine shrimp Artemia sp. and the rotifer Brachionus plicatilis. Swimming speed was investigated as a behavioural end-point for application in ecotoxicology studies. A first experiment to analyse the linear swimming speed of the two organisms was performed to verify the applicability of the video-camera tracking system, here referred to as Swimming Behavioural Recorder (SBR). A second experiment was performed, exposing organisms to different toxic compounds (zinc pyrithione, Macrotrol MT-200, and Eserine). Swimming speed alteration was analyzed together with mortality. The results of the first experiment indicate that SBR is a suitable tool to detect linear swimming speed of the two organisms, since the values have been obtained in accordance with other studies using the same organisms (3.05 mm s(-1) for Artemia sp. and 0.62 mm s(-1) for B. plicatilis). Toxicity test results clearly indicate that swimming speed of Artemia sp. and B. plicatilis is a valid behavioural end-point to detect stress at sub-lethal toxic substance concentrations. Indeed, alterations in swimming speed have been detected at toxic compound concentrations as low as less then 0.1-5% of their LC(50) values. In conclusion, the SSA test with B. plicatilis and Artemia sp. can be a good behavioural integrated output for application in marine ecotoxicology and environmental monitoring programs.

Characterization of Esterases Produced by a Ruminal Bacterium Identified as Butyrivibrio fibrisolvens1

PubMed Central

Lanz, Wayne W.; Williams, Phletus P.

1973-01-01

An obligately anaerobic ruminal bacterial isolate was selected from 18 tributyrin-degrading isolates and identified as Butyrivibrio fibrisolvens strain 53. The culture in late exponential phase contained enzymes which could be released by sonic disruption. These enzymes degraded substrates at a rate in the order 1-naphthyl acetate (NA) > 1-naphthyl butyrate > 1-naphthyl propionate but did not degrade 1-naphthyl palmitate or 1-naphthyl phosphate. The enzymes on NA were neither stimulated nor inhibited by CoCl2, MgCl2, and MnCl (each varied from 10−6 to 10−4 M). CaCl at 10−3 M stimulated esterase activity by 16%. Aliphatic substrates were hydrolyzed at a rate in the order triacetin > tributyrin > tripropionin, and ethyl acetate > ethyl formate. Similarly, aromatic fluorescein diesters were degraded at a rate in the order acetyl > propionyl > caproyl > butyryl > capryl > lauryl. Polyacrylamide gel electrophoretic zymograms indicated that the enzyme composite contained cathodally migrating bands. By column chromatography, these enzymes were separated into six NA-degrading fractions. Fraction V contained an esterase which had an optimal temperature of 39 C, a Km of 7.6 × 10−4 on NA, and a molecular weight of about 66,000. This enzyme was inhibited by paraoxon (41%, 10−4 M), eserine (17%, 10−2 M), NaF (17%, 10−2 M), and diisopropyl fluorophosphate (62%, 10−4 M) but not by 1-naphthyl N-methyl carbamate at 8.4 × 10−4 M. PMID:4734862

Sound radiation and wing mechanics in stridulating field crickets (Orthoptera: Gryllidae).

PubMed

Montealegre-Z, Fernando; Jonsson, Thorin; Robert, Daniel

2011-06-15

Male field crickets emit pure-tone mating calls by rubbing their wings together. Acoustic radiation is produced by rapid oscillations of the wings, as the right wing (RW), bearing a file, is swept across the plectrum borne on the left wing (LW). Earlier work found the natural resonant frequency (f(o)) of individual wings to be different, but there is no consensus on the origin of these differences. Previous studies suggested that the frequency along the song pulse is controlled independently by each wing. It has also been argued that the stridulatory file has a variable f(o) and that the frequency modulation observed in most species is associated with this variability. To test these two hypotheses, a method was developed for the non-contact measurement of wing vibrations during singing in actively stridulating Gryllus bimaculatus. Using focal microinjection of the neuroactivator eserine into the cricket's brain to elicit stridulation and micro-scanning laser Doppler vibrometry, we monitored wing vibration in actively singing insects. The results show significantly lower f(o) in LWs compared with RWs, with the LW f(o) being identical to the sound carrier frequency (N=44). But during stridulation, the two wings resonate at one identical frequency, the song carrier frequency, with the LW dominating in amplitude response. These measurements also demonstrate that the stridulatory file is a constant resonator, as no variation was observed in f(o) along the file during sound radiation. Our findings show that, as they engage in stridulation, cricket wings work as coupled oscillators that together control the mechanical oscillations generating the remarkably pure species-specific song.

Direct Measurement of Acetylesterase in Living Protist Cells1

PubMed Central

Medzon, Edward L.; Brady, Marilyn L.

1969-01-01

The fluorogenic acetylesterase (acetic ester hydrolase EC 3.1.1.6.) substrate, fluorescein diacetate, was used to measure enzyme activity in living protist cells. The visual enzyme assay was done by monitoring fluorochromasia by fluorescent microscopy. Quantitative fluorogenic assays were done by measuring the evolved fluorescein in a fluorometer. Of 59 strains of bacteria, 35 were fluorochromatically positive. Eight of the fluorochromatically negative strains were fluorogenically positive. Of 22 strains of slime molds and fungi, all were fluorochromatically positive. Three out of 12 different algae were fluorochromatically positive. Several unidentified protozoa were also fluorochromatically positive. Four out of six protozoa were fluorochromatically positive. Structures of special interest showing acetylesterase activity were: the growing hyphal tips of fungi, the vacuolated areas of yeast and protozoa, newly formed bacterial spores or immature fungal spores, “mesosome-like” bodies in Bacillus megaterium, and the cell membrane and nuclear region of green algae. Yeast protoplasts and bacterial protoplasts and spheroplasts were fluorochromatically positive when derived from positive cells and negative when derived from negative cells. There was no correlation between the possession of a capsule and acetylesterase activity. There was no effect on the viability of bacterial cells incubated in the presence of fluorescein diacetate. Paraoxon inhibited bacterial and yeast enzyme at 10−5m. Eserine (10−5m) and Paraoxon (10−7m) inhibited B. megaterium enzyme. Sodium acetate at 10−2m did not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed. Images PMID:4974398

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

PubMed

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

2001-06-01

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

Characterization of plasma cholinesterase from the White stork (Ciconia ciconia) and its in vitro inhibition by anticholinesterase pesticides.

PubMed

Oropesa, Ana-Lourdes; Gravato, Carlos; Sánchez, Susana; Soler, Francisco

2013-11-01

Blood plasma cholinesterase (ChE) activity is a sensitive biomarker of exposure to organophosphorus (OP) and carbamate (CB) insecticides in vertebrates. Several studies indicate that more than one ChE form may be present in blood of birds. In this study the predominant ChE activity (acetylcholinesterase - AChE- or butyrylcholinesterase - BChE-), the range of ChE activity as well as ChE age-dependent changes in non-exposed individuals of White stork (Ciconia ciconia) have been established. The in vitro sensitivity of ChE to OP and CB insecticides such as paraoxon-methyl, carbofuran and carbaryl was also investigated. Plasma ChE was characterised using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51 and iso-OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma cholinesterase followed by acetylcholine and butyrylcholine and the predominant enzymatic activity in plasma of White storks was BChE. Normal plasma BChE activity in White stork was 0.32±0.01μmol/min/ml for adults and 0.28±0.03μmol/min/ml for juveniles. So, the age had no significant effect on the range of BChE activity. The study on the in vitro inhibitory potential of tested anticholinesterase pesticides on plasma ChE activity revealed that paraoxon-methyl is the most potent inhibitor followed by carbofuran and finally by carbaryl. The percentage of in vitro plasma ChE inhibition was observed to be similar between adults and juveniles. Copyright © 2013 Elsevier Inc. All rights reserved.

Cholinesterase activity in the tissues of bivalves Noah's ark shell (Arca noae) and warty venus (Venus verrucosa): characterisation and in vitro sensitivity to organophosphorous pesticide trichlorfon.

PubMed

Perić, Lorena; Ribarić, Luka; Nerlović, Vedrana

2013-08-01

Cholinesterase (ChE, EC 3.1.1.7) activity was investigated in gills and adductor muscle of two bivalve species: Arca noae and Venus verrucosa. The properties of ChEs were investigated using acetylcholine iodide (ASCh), butyrylcholine iodide (BSCh) and propionylcholine iodide (PrSCh) as substrates and eserine, BW254c51 and iso-OMPA as specific inhibitors. The highest level of ChE activity in crude tissue extracts was detected with PrSCh followed by ASCh, while values obtained with BSCh were apparently low, except in A. noae adductor muscle. The enzyme activity in A. noae gills and V. verrucosa gills and adductor muscle was significantly inhibited by BW254c51, but not with iso-OMPA. ChE activity in adductor muscle of A. noae was significantly reduced by both diagnostic inhibitors. The effect of organophosphorous pesticide trichlorfon on ChE activity was investigated in vitro in both species as well as in the gills of mussels Mytilus galloprovincialis. The highest sensitivity of ChE to trichlorfon was observed in A. noae gills and adductor muscle (IC50 1.6×10(-7)M and 1.1×10(-7)M, respectively), followed by M. galloprovincialis gills (IC50 1.0×10(-6)M) and V. verrucosa gills and adductor muscle (IC50 1.7×10(-5)M and 0.9×10(-5)M, respectively). The results of this study suggest the potential of ChE activity measurement in the tissues of A. noae as effective biomarker of OP exposure in marine environment. Copyright © 2013 Elsevier Inc. All rights reserved.

Presynaptic Muscarinic M2 Receptors Modulate Glutamatergic Transmission in the Bed Nucleus of the Stria Terminalis

PubMed Central

Guo, Ji-Dong; Hazra, Rimi; Dabrowska, Joanna; Muly, E. Chris; Wess, Jürgen; Rainnie, Donald G.

2012-01-01

The anterolateral cell group of the bed nucleus of the stria terminalis (BNSTALG) serves as an important relay station in stress circuitry. Limbic inputs to the BNSTALG are primarily glutamatergic and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Significantly, local infusion of acetylcholine (ACh) receptor agonists into the BNST trigger stress-like cardiovascular responses, however, little is known about the effects of these agents on glutamatergic transmission in the BNSTALG. Here, we show that glutamate- and ACh-containing fibers are found in close association in the BNSTALG. Moreover, in the presence of the acetylcholinesterase inhibitor, eserine, endogenous ACh release evoked a long-lasting reduction of the amplitude of stimulus-evoked EPSCs. This effect was mimicked by exogenous application of the ACh analogue, carbachol, which caused a reversible, dose-dependent, reduction of the evoked EPSC amplitude, and an increase in both the paired pulse ratio and coefficient of variation, suggesting a presynaptic site of action. Uncoupling of postsynaptic G-proteins with intracellular GDP-β-S, or application of the nicotinic receptor antagonist, tubocurarine, failed to block the carbachol effect. In contrast, the carbachol effect was blocked by prior application of atropine or M2 receptor-preferring antagonists, and was absent in M2/M4 receptor knockout mice, suggesting that presynaptic M2 receptors mediate the effect of ACh. Immuno-electron microscopy studies further revealed the presence of M2 receptors on axon terminals that formed asymmetric synapses with BNST neurons. Our findings suggest that presynaptic M2 receptors might be an important modulator of the stress circuit and hence a novel target for drug development. PMID:22166222

A central site of action for benzamide facilitation of gastric emptying.

PubMed

Costall, B; Gunning, S J; Naylor, R J; Simpson, K H

1983-07-22

Gastric emptying of the fed guinea-pig was measured using a non-invasive X-ray fluoroscopic technique to determine passage from the stomach of polystyrene-coated barium sulphate spheroids. Peripherally administered metoclopramide (0.1-10 mg/kg i.p.), clebopride (1-10 mg/kg i.p.), (-)-sulpiride (40 mg/kg i.p.), haloperidol (1 mg/kg i.p.) and domperidone (1-10 mg/kg i.p.) failed to modify gastric emptying. Stress inhibited emptying, and this was considered to explain the effects of eserine and high dose metoclopramide. Gastric emptying was decreased by peripherally administered atropine (0.5 mg/kg i.p.) and apomorphine (0.1-0.5 mg/kg s.c.); the apomorphine response was antagonised by pretreatment with haloperidol, domperidone, (-)-sulpiride, metoclopramide and clebopride but not by prazosin + propranolol. Gastric emptying was facilitated by intracerebroventricular (i.c.v.) administrations of metoclopramide and clebopride (40, 100 and 200 micrograms) but not by i.c.v. domperidone, haloperidol, fluphenazine or (-)-sulpiride (100, 200 micrograms) and was inhibited by i.c.v. apomorphine (100, 200 micrograms); the response to i.c.v. apomorphine was antagonised by i.c.v. pretreatments with haloperidol, domperidone, (-)-sulpiride, metoclopramide and clebopride (40-50 micrograms). Facilitation of emptying by i.c.v. metoclopramide was prevented by peripheral pretreatment with atropine (0.5 mg/kg i.p.). It is concluded that the actions of apomorphine and metoclopramide/clebopride to respectively inhibit and facilitate gastric emptying may be mediated, at least in part, via central mechanisms. Whilst apomorphine's action may be mediated via dopamine receptor mechanisms, metoclopramide and clebopride act at additional unspecified sites, metoclopramide's action being expressed via cholinergic mechanisms.

Variability of esterase patterns in adult flies of the saltans species group of Drosophila (subgenus Sophophora).

PubMed

Bernardo, Alessandra Augusta; Bicudo, Hermione Elly Melara de Campos

2009-09-01

Esterases are known for their involvement in several physiological processes and high degree of polymorphism, in many organisms. Such polymorphism has been used to characterize species and species groups and to study genetic changes occurred in their evolutionary history. In the present study, the esterase patterns of 19 strains from 10 species representative of the five subgroups of the saltans species group were analyzed using polyacrylamide gel electrophoresis and alpha- and beta- naphthyl acetates as substrates. Fifty-one esterase bands were detected and classified as 31 alpha-esterases, 18 beta-esterases and two alpha/beta-esterases. On the basis of the inhibition patterns using Malathion and eserine sulfate, 34 bands were classified as carboxylesterases, 14 as acethylesterases and three as cholinesterases. Ten gene loci were tentatively established on the basis of data on band position in the gel, substrate preference and inhibition pattern. Twenty bands were species-specific, the remaining being shared by species from the same or different subgroups. Bands detected exclusively in males and bands with a different frequency or degree of expression between sexes were also detected. In the gels prepared for analysis of gene expression in the body parts (head, thorax and abdomen), the degree of expression of the beta-esterases was higher in the thorax, while the alpha-esterases were expressed predominantly in the abdomen and thorax. A global view of the data available at present on the esterases of the species from the saltans group and their degree of polymorphism are presented, as well as the possibility of using some beta-esterases, because of their characteristics in the gels, as markers for species identification.

Characterization of plasma cholinesterase in rabbit and evaluation of the inhibitory potential of diazinon.

PubMed

Oropesa, Ana Lourdes; Pérez-López, Marcos; Soler, Francisco

2014-02-01

Several studies indicate that more than one cholinesterase form may be present in the blood of mammals. In this study the predominant plasma cholinesterase activity, the physiological cholinesterase activity as well as cholinesterase sex-dependent changes in non-exposed individuals of rabbit have been established. Plasma cholinesterase was characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three cholinesterase inhibitors (eserine sulfate, BW284C51 and iso-OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma cholinesterase followed by acetylthiocholine and butyrylthiocholine, and the predominant enzymatic activity was acetylcholinesterase. Physiological plasma cholinesterase activity was 198.9 ± 5.8 nmol/min/ml for male and 205.2 ± 5.0 nmol/min/ml for female using acetylthiocholine as substrate. Thus, sex had no significant effect on the physiological cholinesterase activity (p>0.05). In addition, the in vivo and in vitro sensitivity of plasma cholinesterase to diazinon was also investigated. In rabbits exposed to single doses of diazinon (25 or 125 mg/kg) the higher inhibitions of plasma cholinesterase were reached 9h after oral administration (53% and 87% inhibition, respectively). Cholinesterase activity significantly recovered up to values similar to pre-administration between 3 and 7d depending on the administered dose and sex of the animals. Plasma cholinesterase activity decreased to 24%, 53% and 74% of the initial activity at 9h of in vitro exposure to 1.25, 3.13 and 6.25mg/l of diazinon, respectively, and it remained steadily depressed throughout the experimental period (10d). This study has demonstrated the sensitivity of cholinesterase activity in plasma of rabbits following both in vivo and in vitro exposure to sub-lethal concentrations of diazinon. © 2013 Published by Elsevier Inc.

Evidence that exercise-induced heat storage is dependent on adrenomedullary secretion.

PubMed

Rodrigues, A G; Lima, N R V; Coimbra, C C; Marubayashi, U

2008-06-09

To investigate the influence of medullary adrenal secretion on thermoregulation during exercise, Phy (Eserine, 5x10(-3) M) was injected into the lateral cerebral ventricle of normal (INT) or bilaterally adrenodemedullated (ADM) untrained rats. Body temperature (Tb) and metabolic rate were measured in the rats while they were exercising on a treadmill (20 m min(-1), 5% inclination) until fatigue or while they were at rest after drug injection. In resting rats, Phy increased oxygen consumption in both INT or ADM rats without any effect on core temperature. During the dynamic phase of exercise (first 20 min), ADM attenuated the exercise-induced increase in core temperature (0.86+/-0.12 degrees C ADM Sal vs 1.48+/-0.21 degrees C INT Sal), thus reducing heat storage (HS) levels. Icv injection of Phy in ADM rats significantly reduced the increase in Tb (0.012+/-0.10 degrees C min(-1) Phy vs 0.042+/-0.006 degrees C min(-1) Sal; p<0.02) and HS (65.8+/-56.1 cal Phy vs 207.7+/-32.7 cal Sal; p<0.04) compared to ADM Sal rats. In conclusion, the exercise-induced increase in heat storage was attenuated by adrenodemedullation in rats. Furthermore, the activation of heat loss mechanisms by the central cholinergic system during exercise occurs independently of adrenal medullary secretion suppression and can be improved by previous adrenodemedullation. Our data indicate the existence of a dual mechanism of heat loss control during the dynamic phase of exercise: one involving sympathoadrenal system activation that impairs heat loss and another that counteracts the increased sympathoadrenal activity through the hypothalamic cholinergic system to promote heat loss.

Characterization of ML0314c of Mycobacterium leprae and deciphering its role in the immune response in leprosy patients.

PubMed

Kaur, Gurkamajit; Sharma, Aashish; Narang, Tarun; Dogra, Sunil; Kaur, Jagdeep

2018-02-15

Mycobacterium leprae has a reduced genome size due to the reductive evolution over a long period of time. Lipid metabolism plays an important role in the life cycle and pathogenesis of this bacterium. In comparison to 26 lip genes (Lip A-Z) of M. tuberculosis, M. leprae retained only three orthologs indicating their importance in its life cycle. ML0314c (LipU) is one of them. It is conserved throughout the mycobacterium species. Bioinformatics analysis showed the presence of an α/β hydrolase fold and 'GXSXG' characteristic of the esterases/lipases. The gene was expressed in E. coli and purified to homogeneity. It showed preference towards short chain esters with pNP-acetate as the preferred substrate. The enzyme showed optimal activity at 45°C and pH8.0. ML0314c protein was stable between temperatures ranging from 20 to 60°C and pH5.0-8.0, i.e., relatively acidic and neutral conditions. The active site residues predicted bioinformatically were confirmed to be Ser168, Glu267, and His297 by site directed mutagenesis. E-serine, DEPC and Tetrahydrolipstatin (THL) completely inhibited the activity of ML0314c. The protein was localized in cell wall and extracellular medium. Several antigenic epitopes were predicted in ML0314c. Protein elicited strong humoral immune response in leprosy patients, whereas, a reduced immune response was observed in the relapsed cases. No humoral response was observed in treatment completed patients. Overexpression of ml0314c in the surrogate host M. smegmatis showed marked difference in the colony morphology and growth rate. In conclusion, ML0314c is a secretary carboxyl esterase that could modulate the immune response in leprosy patients. Copyright © 2017 Elsevier B.V. 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 negatively correlated sensitivity, being insensitive to phosphamidon and monocrotophos and sensitive to N -methyl carbamates. PMID:25373165

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: 1,1-diphenyl-2-picrylhydrazyl, Cd: Corchorus depressus, AChE: Acetylcholinesterase, BChE: Butyrylcholinesterase, AD: Alzheimer's disease. PMID:29200727

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization.

PubMed

Chandrashekharaiah, K S; Swamy, N Ramachandra; Murthy, K R Siddalinga

2011-12-01

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS-PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS-PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K(m) of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K(m) was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45°C (ME-III) and 37°C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35°C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB). Copyright © 2011 Elsevier Ltd. All rights reserved.

Enzymes mediating resistance to lambda-cyhalothrin in Eriopis connexa (Coleoptera: Coccinellidae).

PubMed

Rodrigues, Agna R S; Siqueira, Herbert A A; Torres, Jorge B

2014-03-01

Resistance to widely used insecticide, lambda-cyhalothrin, was recently reported in the predatory lady beetle Eriopis connexa (Germar) (Coleoptera: Coccinellidae). However, to understand whether metabolic mechanisms underlie such resistance, synergism bioassays and in vitro studies were carried out by using inhibitors and model substrates for enzymatic assays, respectively. The LD50s estimated for susceptible and resistant populations ηg of lambda-cyhalothrin/insect, and thus, a 22-fold difference in resistance ratio. Synergism ratios for the susceptible population with piperonyl butoxide (PBO), diethyl maleate (DEM), triphenyl phosphate (TPP), and S,S,S-tributylphosphorotrithioate (DEF) were respectively 33.8-, 0.24-, 0.35-, and 4.25-fold, while for the resistant population, they were 1463.0-, 0.79-, 0.85-, and 282.6-fold, respectively. The synergized resistance ratios were 0.50-, 2.00-, 6.75-, and 8.77-fold with PBO, DEF, DEM, and TPP, respectively, while resistance was virtually suppressed with DEF. The esterase exhibited 4.16-, 4.03-, and 5.38-fold greater activity towards formation of α-naphthol, β-naphthol, and 4-nitrophenol in the resistant population of E. connexa than in the susceptible population. The activity of esterase depended on concentrations of DEF applied, either using α-naphthol or β-naphthol, which completely inhibited the activity at 636 ηM. The PBO inhibited the β-naphthol formation in approximately 50%, suggesting it as inhibitor of esterases. The activities of glutathione-S-transferase were similar and corresponded to 0.36-0.47 ηmol(-1) min(-1)μg of protein, for S and R populations, respectively. Similarly, the activities of cytochrome P450-dependent microsomal monooxygenases were 0.04 and 0.05 ηmol(-1) min(-1)μg of protein. The native gel indicated that the formation of β-naphthol was completely inhibited by methyl-paraoxon, but only partially inhibited by eserine, TPP, and PBO. Although other studies with DEF and PBO have demonstrated strong inhibition of type B carboxylesterase associated with insecticide resistance, the results reported here do not rule out metabolism by cytochrome P450-dependent microsomal monooxygenases as a factor conferring E. connexa resistance to lambda-cyhalothrin and confirmed that PBO may also act by inhibiting esterases of insects. Copyright © 2014 Elsevier Inc. All rights reserved.

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 Strigidae families were characterized by a BChE contribution that dominated the total ChE activity, while in the species of the Falconidae family, AChE activity dominated. With the exception of the barn owl, CbE activity (eserine-insensitive alpha-naphthyl acetate esterase [alpha-NAE] activity) in all species was almost absent or very low. The values obtained in this study for ChE, AChE, and BChE activities and the AChE:BChE ratios for buzzard, kestrel, barn owl, and tawny owl provide a good estimate of the normal values in free-living individuals of these European species. They can be used as a baseline to evaluate the effect of anticholinesterase insecticides in the field.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties

PubMed Central

2013-01-01

Background Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. Methods A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3’-5’-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman’s assay in microplates. Results A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P. papatasi AChE1 was expressed in the baculovirus system and characterized as an insect acetylcholinesterase with substrate preference for acetylthiocholine and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine, BW284c51, malaoxon, and paraoxon, and was insensitive to the butyrylcholinesterase inhibitors ethopropazine and iso-OMPA. Conclusions Results presented here enable the screening and identification of PpAChE mutations resulting in the genotype for insensitive PpAChE. Use of the recombinant P. papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition. PMID:23379291

The combined effects of pyridostigmine and chronic stress on brain cortical and blood acetylcholinesterase, corticosterone, prolactin and alternation performance in rats.

PubMed

Kant, G J; Bauman, R A; Feaster, S R; Anderson, S M; Saviolakis, G A; Garcia, G E

2001-01-01

Thousands of soldiers who served in the Gulf War have symptoms that have been collectively termed Gulf War Illness (GWI). It has been suggested that a combination of operational stress and pyridostigmine, a drug given as a pretreatment to protect soldiers against the effects of exposure to nerve agents, might have had unexpected adverse health effects causing these symptoms. Our laboratory has previously modeled operational stress in rats using a paradigm of around-the-clock intermittent signalled footshock. In the present studies, this model was used to investigate the potential synergistic effects of chronic stress and pyridostigmine on physiology and behavior. Seventy-two rats were trained to perform an alternation lever pressing task to earn their entire daily food intake. The rats were then implanted with osmotic minipumps containing vehicle, pyridostigmine (25 mg/ml pyridostigmine bromide) or physostigmine (20 mg/ml eserine hemisulfate). The pumps delivered 1 microl/h, which resulted in a cumulative dosing of approximately 1.5 mg/kg/day of pyridostigmine or 1.2 mg/kg/day of physostigmine, equimolar doses of the two drugs. The rats were then returned to their home cages where performance continued to be measured 24 h/day. After 4 days, 24 of the 72 rats were trained to escape signalled footshock (avoidance-escape group) and 24 other rats (yoked-stressed group) were each paired to a rat in the avoidance-escape group. The remaining 24 rats were not subjected to footshock (unstressed group). Shock trials were intermittently presented in the home cage 24 h/day for 3 days, while alternation performance continued to be measured. Since only 12 test cages were available, each condition was repeated to achieve a final n of six rats per group. Pyridostigmine and physostigmine each decreased blood acetylcholinesterase levels by approximately 50%. Physostigmine also decreased brain cortical acetylcholinesterase levels by approximately 50%, while pyridostigmine had no effect on cortical acetylcholinesterase activity. Alternation performance was impaired on the first day of stress and then recovered. Neither pyridostigmine nor physostigmine affected performance in the absence of stress or increased the effects of stress alone. Corticosterone was significantly increased in the yoked stress group compared to unstressed controls. These data suggest that pyridostigmine does not exacerbate the effects of stress on performance or levels of stress hormones. Furthermore, these data do not suggest that stress enables pyridostigmine to cross the blood brain barrier.

Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases.

PubMed

Bianco, Karina; Yusseppone, María Soledad; Otero, Sofía; Luquet, Carlos; Ríos de Molina, María Del Carmen; Kristoff, Gisela

2013-11-15

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 μg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 μg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 μg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 μg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 μg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile. Copyright © 2013 Elsevier B.V. All rights reserved.

Resistance in cholinesterase activity after an acute and subchronic exposure to azinphos-methyl in the freshwater gastropod Biomphalaria straminea.

PubMed

Bianco, Karina; Otero, Sofía; Oliver, Agustina Balazote; Nahabedian, Daniel; Kristoff, Gisela

2014-11-01

Organophosphorous and carbamates insecticides are ones of the most popular classes of pesticides used in agriculture. Its success relies on their high acute toxicity and rapid environmental degradation. These insecticides inhibit cholinesterase and cause severe effects on aquatic non-target species, particularly in invertebrates. Since the properties of cholinesterases may differ between species, it is necessary to characterize them before their use as biomarkers. Also organophosphorous and carbamates inhibit carboxylesterases and the use of both enzymes for biomonitoring is suggested. Azinphos-methyl is an organophosphorous insecticide used in several parts of the word. In Argentina, it is the most applied insecticide in fruit production in the north Patagonian region. It was detected with the highest frequency in superficial and groundwater of the region. This work aims to evaluate the sensitivity of B. straminea cholinesterases and carboxylesterases to the OP azinphos-methyl including estimations of 48 h NOEC and IC50 of the pesticide and subchronic effects at environmentally relevant concentrations. These will allow us to evaluate the possibility of using cholinesterase and carboxylesterase of B. straminea as sensitive biomarkers. Previously a partial characterization of these enzymes will be performed. As in most invertebrates, acetylthiocholine was the preferred hydrolyzed substrate of B. straminea ChE, followed by propionylthiocholine and being butyrylthiocholine hydrolysis very low. Cholinesterase activity of B. straminea was significantly inhibited by the selective cholinesterases inhibitor (eserine) and by the selective inhibitor of mammalian acethylcholinesterase (BW284c51). In contrast, iso-OMPA, a specific inhibitor of butyrylcholinesterase, did not inhibit cholinesterase activity. These results suggest that cholinesterase activity in total soft tissue of B. straminea corresponds to acethylcholinesterase. Carboxylesterases activity was one order of magnitude higher than cholinesterase. A greater efficiency (Vmax/Km) was obtained using acetylthiocholine and p-nitrophenyl butyrate. Acute exposure to azinphos-methyl did not cause inhibition of cholinesterase activity until 10 mg L(-1) used. Carboxylesterases towards p-nitrophenyl butyrate was inhibited by azinphos-methyl being the IC502.20±0.75 mg L(-1) of azinphos-methyl. Subchronic exposure to environmental concentrations of azinphos-methyl (0.02 and 0.2 mg L(-1)) produced a decrease in survival, protein content and carboxylesterases activity despite no inhibition of cholinesterase activity was observed. B. straminea cholinesterase is not a sensible biomarker. On the contrary, carboxylesterases activity was inhibited by azinphos-methyl. Carboxylesterases could be protecting cholinesterase activity and therefore, protecting the organism from neurotoxicity. This work confirms the advantages of measuring cholinesterases and carboxylesterases jointly in aquatic biomonitoring of pesticide contamination. This becomes relevant in order to find more sensitive biomarkers and new strategies to protect non-target aquatic organisms from pesticide contamination. Copyright © 2014 Elsevier Inc. All rights reserved.

Pharmacology of pH effects on carotid body chemoreceptors in vitro.

PubMed

Eyzaguirre, C; Zapata, P

1968-04-01

1. The carotid body and the carotid nerve were removed from anaesthetized cats and placed in a small Perspex channel through which Locke solution (at various pH values and usually equilibrated with 50% O(2) in N(2)) was allowed to flow. The glomus was immersed in the flowing solution while the nerve was lifted into oil covering the saline. Sensory discharges were recorded from the nerve and their frequency was used as an index of receptor activity. At times, a small segment of carotid artery, containing pressoreceptor endings, was removed together with the glomus. In this case, pressoreceptor discharges were recorded from the nerve.2. The amplitude of either chemo- or pressoreceptor discharges was not changed by strong acid solutions. Acid decreased the frequency of the baroreceptor discharges only when pH fell to less than 4.0. Solutions at low pH increased the chemosensory discharge, but acid depressed the increased chemoreceptor discharge elicited by KCl. These experiments indicated that H(+) ions probably acted as membrane ;stabilizers' without depolarizing either the nerve fibres or endings.3. Acid solutions increased the action of acetylcholine chloride (AChCl) (100-200 mug) on chemoreceptors. This effect probably was due either to inactivation of tissue cholinesterase or to enhanced sensitivity of the sensory endings to ACh.4. Choline chloride (10(-3)M), which favours ACh synthesis, protected the preparation against decay during prolonged experimentation. Hemicholinium-3 (HC-3), which blocks ACh synthesis in low concentrations (10(-5)M), depressed the chemosensory response to acid and to hypoxia when such stimuli were applied repeatedly. This concentration of HC-3 did not change effects of applied ACh.5. Substances which affect ACh release markedly changed the chemoreceptor discharge increase induced by acidity and other forms of stimulation. In the absence of Ca(2+), acid, anoxia, and interruption of flow provoked receptor depression while receptor excitation induced by ACh and KCl persisted. All stimuli excited and showed increased effectiveness as the Ca(2+) concentration was raised, but their effects declined as Ca(2+) was increased above normal values. Mg(2+) ions depressed the chemoreceptor effects induced by all these stimuli. The action of Mg(2+) was not due entirely to nerve ending block. Morphine sulphate (which decreases ACh release in other structures) also depressed the receptor response to acid and flow interruption.6. Cholinergic blocking agents such as mecamylamine, hexamethonium, atropine, dihydro-beta-erithroidine (DHE), HC-3 (10(-4)M), choline and acetylcholine (in combination with choline) depressed the effects of acid and ACh on the chemoreceptors. The effect induced by interruption of flow was depressed only by mecamylamine and DHE.7. Agents which affect the fate of released ACh, such as acetylcholinesterase and eserine salicylate, did not affect clearly the response of chemoreceptors to acid.8. The results suggest that acid stimulates chemoreceptor fibres through an indirect mechanism, viz. through increased release and/or decreased destruction of a presynaptic transmitter from the glomus cell. This transmitter is probably ACh (see following paper, Eyzaguirre & Zapata, 1968).

Pharmacology of pH effects on carotid body chemoreceptors in vitro

PubMed Central

Eyzaguirre, C.; Zapata, P.

1968-01-01

1. The carotid body and the carotid nerve were removed from anaesthetized cats and placed in a small Perspex channel through which Locke solution (at various pH values and usually equilibrated with 50% O2 in N2) was allowed to flow. The glomus was immersed in the flowing solution while the nerve was lifted into oil covering the saline. Sensory discharges were recorded from the nerve and their frequency was used as an index of receptor activity. At times, a small segment of carotid artery, containing pressoreceptor endings, was removed together with the glomus. In this case, pressoreceptor discharges were recorded from the nerve. 2. The amplitude of either chemo- or pressoreceptor discharges was not changed by strong acid solutions. Acid decreased the frequency of the baroreceptor discharges only when pH fell to less than 4·0. Solutions at low pH increased the chemosensory discharge, but acid depressed the increased chemoreceptor discharge elicited by KCl. These experiments indicated that H+ ions probably acted as membrane `stabilizers' without depolarizing either the nerve fibres or endings. 3. Acid solutions increased the action of acetylcholine chloride (AChCl) (100-200 μg) on chemoreceptors. This effect probably was due either to inactivation of tissue cholinesterase or to enhanced sensitivity of the sensory endings to ACh. 4. Choline chloride (10-3 M), which favours ACh synthesis, protected the preparation against decay during prolonged experimentation. Hemicholinium-3 (HC-3), which blocks ACh synthesis in low concentrations (10-5 M), depressed the chemosensory response to acid and to hypoxia when such stimuli were applied repeatedly. This concentration of HC-3 did not change effects of applied ACh. 5. Substances which affect ACh release markedly changed the chemoreceptor discharge increase induced by acidity and other forms of stimulation. In the absence of Ca2+, acid, anoxia, and interruption of flow provoked receptor depression while receptor excitation induced by ACh and KCl persisted. All stimuli excited and showed increased effectiveness as the Ca2+ concentration was raised, but their effects declined as Ca2+ was increased above normal values. Mg2+ ions depressed the chemoreceptor effects induced by all these stimuli. The action of Mg2+ was not due entirely to nerve ending block. Morphine sulphate (which decreases ACh release in other structures) also depressed the receptor response to acid and flow interruption. 6. Cholinergic blocking agents such as mecamylamine, hexamethonium, atropine, dihydro-β-erithroidine (DHE), HC-3 (10-4 M), choline and acetylcholine (in combination with choline) depressed the effects of acid and ACh on the chemoreceptors. The effect induced by interruption of flow was depressed only by mecamylamine and DHE. 7. Agents which affect the fate of released ACh, such as acetylcholinesterase and eserine salicylate, did not affect clearly the response of chemoreceptors to acid. 8. The results suggest that acid stimulates chemoreceptor fibres through an indirect mechanism, viz. through increased release and/or decreased destruction of a presynaptic transmitter from the glomus cell. This transmitter is probably ACh (see following paper, Eyzaguirre & Zapata, 1968). PMID:4296975