Showed Acetylcholinesterase Activity Research Articles

Synthesis and Evaluation of Biological Activity of New Arylphosphoramidates.

The synthesis of new substituted arylphosphoramidates is performed in two steps through phosphorylation of the corresponding alcohols followed by aminolysis. The formation of the desired phosphoramidates depends on the subsequent addition of the two alcohols with the amine being added at the last step. The products were obtained in 58–95% yields. They were characterized mainly by multinuclear (1H, 13C, 31P, and 19F) NMR and IR spectroscopy. In addition, the antimicrobial and antiacetylcholinesterase activities were evaluated. The results showed acetylcholinesterase activity by some compounds, whilst no significant inhibitory effect against the tested bacterial strains has been recorded.

Molecular characterization of Hydra acetylcholinesterase and its catalytic activity

A full-length cDNA encoding an acetylcholinesterase (AChE) from Hydra magnipapillata was isolated. All of the important aromatic residues that line a catalytic gorge in cholinesterases of other species were conserved, but the sequences of peripheral anionic and choline binding sites were not. Hydra AChE, expressed in Xenopus oocytes, showed AChE activity. The gene was expressed in both ectodermal and endodermal epithelial cells except for the tentacles and basal disk. AChE gene expression was not detected in the regenerating tips in either the head or the foot, indicating that regeneration is controlled by the non-neuronal cholinergic system in Hydra.

Relation between pesticide exposure and intrauterine growth retardation

The increased use of organophosphorus insecticides in agriculture and their widespread existence in the environment poses a potential health hazard. To determine the relationship between exposure to pesticides and intrauterine growth retardation (IUGR), live newborns from singleton pregnancies, with ( n=79) and without ( n=292) IUGR were studied. During the gestational period the mothers were living in agricultural communities in the state of Chihuahua, Mexico. Exposure to agrochemical products was evaluated. A significant association between the history of positive exposure to pesticides (i.e. the women themselves or their newborns who showed acetylcholinesterase activity levels lower than 20%) and the presence of IUGR was found. The proportions of exposure in the cases were 18% and 8% in the control group; the adjusted OR (fat free mass, anti-cytomegalovirus antibodies and placental weight) was 2.33 ( p=0.04).

Excitatory amino acid-induced currents in rat septal cholinergic neurons in culture.

Whole-cell voltage-clamp recordings were used to study excitatory amino acid-induced currents in neurons isolated from the septum of fetal rat brains. The neurons were cultured for more than four weeks on a feeder layer composed of glial cells obtained from the septal region. Septal neurons were either fusiform, triangular or multipolar and 83% of cells showed acetylcholinesterase activity. L-Glutamate, kainate, quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) applied by local perfusion produced inward currents (Iglu, Ikai, Iquis and IAMPA, respectively) at -44mV which increased in amplitude with increasing concentration of agonist; they desensitized when induced at higher concentrations except for the Ikai. The EC50s for the peak Ikai and sustained Iglu, Iquis and IAMPA were 55, 13, 0.39 and 3.5 microM, respectively. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) depressed Ikai and IAMPA evoked at a concentration of 10 microM (IC50s: 0.58 and 0.84 microM, respectively). Schild analysis for the CNQX action on Ikai gave a dissociation constant of 0.27 microM for CNQX. n-Methyl-D-aspartate (NMDA) (with glycine, 3 microM) produced an inward current (INMDA) at -44 mV whose peak amplitude enhanced with increased concentrations (EC50 = 32 microM). INMDA was potentiated by glycine (EC50 = 0.15 microM) and inhibited by D-2-amino-5-phosphovalerate (IC50 = 9.9 microM for INMDA evoked at a concentration of 50 microM). MK-801 (0.1-10 microM) inhibited INMDA in a dose- and use-dependent manner. INMDA was (0.1-10 microM) inhibited INMDA in a dose- and use-dependent manner. INMDA was potentiated by spermine (EC50 = 247 microM; 91% increase at 1mM) in a manner independent of holding potential (VH). INMDA was inhibited by Mg2+ and Zn2+ (IC50 = 673 and 39 microM, respectively, at -44 mV) in a manner dependent on VH; the magnitudes of a depolarization required for an e-fold increase in their IC50s in a range of -64 to -24 mV were 16 and 22 mV, respectively. The action of Zn2+ was independent of VH > -24 mV. Current-voltage relations for Ikai, Iquis and IAMPA exhibited outward rectification, while that of INMDA showed a region of negative conductance at VH < -30 mV, which disappeared in a Mg(2+)-free solution. Reversal potentials for Ikai, Iquis, IAMPA and INMDA were close to 0 mV, indicating the involvement of non-specific cation channels. Increasing extracellular Ca2+ concentration from 2.4 to 30 mM did not affect the Ikai and Iquis, reversal potential showing negligible Ca2+ component, but shifted INMDA reversal potential to a more positive potential, yielding a ratio of Ca2+ permeability to that of monovalent cation to be 13. Cholinergic septal neurons in culture express non-NMDA-(AMPA/kainate-) and NMDA-type of glutamate receptor channels. Their properties were quantitatively similar to those of glutamate receptor channels on other types of neurons in the brain except for the actions of endogenous neuromodulators (Mg2+, Zn2+ and spermine) on NMDA receptor channels. It is suggested that NMDA receptor channels on different types of neurons may play a distinct role depending on a difference in the actions of these neuromodulators.

Intramucosal nerve cells in human small intestine

We investigated the intramucosal nerve cells of the human small intestine with histochemical methods to demonstrate nicotinamide adenine dinucleotide diaphorase and acetylcholinesterase and with morphometry. Intramucosal neurons appeared as solitary cells or in small groups, especially in the ileum. Most intramucosal nerve cell bodies were round or oval; some were flat or spindle-shaped. They mostly lay close to the muscularis mucosae, but some were located within the muscularis mucosae and others were some distance away from it. The processes of some mucosal neurons projected towards the submucosa. Most mucosal nerve cells showed acetylcholinesterase activity. The frequency distribution of nerve cell profile areas in the intramucosal cells in the duodenum differed from that of cells in the ileum ( P < 0.001). There were more large mucosal nerve cells in the mucosa of the duodenum than in the ileum. There was no significant difference between the frequency distributions of cell profile areas of cells of the mucosa and cells of Meissner's and Henle's plexuses in the same region. We conclude that intramucosal nerve cells, similar to those of the submucosal plexus, exist in the human small intestine. The size of intramucosal nerve cell profiles differs between the duodenum and ileum. This is consistent with their possible different functions.

A Histochemical Study of the Distribution of Acetylcholinesterase-Positive Nerves in the Goat Pancreas

The distribution of acetylcholinesterase (AChE)-positive nerve fibers in the goat pancreas was investigated with histochemical methods at the light- and electron-microscopical level. Intrapancreatic ganglia showing AChE activity were found in the interlobular connective tissue or between acini. AChE-positive nerve fibers were distributed in whole glands and formed perivascular and periinsular plexus. Terminals were identified in both the exocrine and the endocrine tissue. These observations make clear that the cholinergic system is involved in the regulation of both the exocrine and endocrine pancreas in the goat.

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.

Differentiation of membrane excitability in isolated cleavage‐arrested blastomeres from early ascidian embryos.

1. Differentiation of excitable cells was studied electrophysiologically and histochemically in cleavage-arrested blastomeres isolated from early ascidian embryos. Blastomeres were isolated at the 4- or 8-cell stage, and cultured in sea water containing cytochalasin B until the time of hatching of control larvae. Electrical responses, immunoreactivity to epidermis-specific monoclonal antibody (2C5) and activity of muscle-specific acetylcholinesterase were examined. 2. All cleavage-arrested blastomeres isolated from an 8-cell embryo differentiated to elicit either muscular- or epidermal-type action potentials, but no neural-type action potentials were observed in these blastomeres. The anterior-animal and the posterior-animal blastomeres developed only epidermal-type action potentials, which involved expression of Ca2+ channels and immunoreactivity to 2C5. One-third of anterior-vegetal blastomeres developed epidermal-type action potentials which are mediated by Ca2+ channels though the immunoreactivity to 2C5 was absent. A majority of remaining blastomeres showed action potentials composed of Ca2+ currents and TEA-sensitive delayed K+ currents (type I response), and a few of them had fast transient K+ currents (A-currents) in addition (type II response). One-third of posterior-vegetal blastomeres developed epidermal-type action potentials without expression of the immunoreactivity to 2C5. The remainder differentiated into muscular-type cells, which expressed Ca2+ currents, TEA-sensitive and TEA-insensitive delayed K+ currents, and showed acetylcholinesterase activity. 3. Cleavage-arrested blastomeres isolated from a 4-cell embryo also differentiated into epidermal- or muscular-type cells, but not neural-type cells. The anterior blastomere, which is the parent cell of anterior-animal and anterior-vegetal blastomeres of an 8-cell embryo, developed epidermal-type, type I or type II responses, as was the case in the anterior-vegetal blastomere isolated from an 8-cell embryo. The posterior blastomere, which was the parent cell of posterior-animal and posterior-vegetal blastomeres of an 8-cell embryo, differentiated into either epidermal-type or muscular-type cells in terms of both membrane excitability and immunochemical reactivity. 4. Cleavage-arrested 1-cell embryos differentiated exclusively into epidermal-type cells in terms of membrane excitability and 2C5 immunoreactivity, even when the cytochalasin B concentration was decreased below 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Histochemical and cytochemical localization of acetylcholinesterase in the cerebellar cortex of the chicken.

The localization of acetylcholinesterase (AChE) was studied in the cerebellar cortex of the crossbred trembler chickens by means of histo- and cytochemical methods. No essential differences between the crossbred normal and the crossbred trembler chickens were observed. The common results were as follows: Under a light microscope AChE activity was predominantly evident in the molecular layer, and secondly in the granular layer. AChE was ultrastructurally distributed principally in the cisternae of rough endoplasmic reticulum (ER) and in a part of nuclear envelope of the Purkinje, the Golgi and some of the basket and granule cells, and in a portion of the sacculus of the Golgi apparatus of the Purkinje cell only. In dendrites and the initial axon of the Purkinje cells the smooth ER also showed AChE activity. Although dendritic terminals of the Golgi cells contained AChE reaction products, the axon terminal did not. Some of the afferent terminal fibers forming the cerebellar glomerulus exhibited weakly a positive AChE reaction, while others in the vicinity did not show any AChE activity at all. However, the enzyme reaction product was localized in the intercellular spaces between a presynaptic afferent terminal and the postsynaptic granule cell dendritic terminals in the glomerulus. In addition, AChE activity was found in the form of spots in the intercellular spaces of both molecular and granular layers.

Mass Isolation of Muscle Lineage Blastomeres from Ascidian Embryos: (ascidian embryos/muscle lineage cells/mass isolation).

The aim of this investigation was to establish an experimental system for studying the causal relationship between DNA replication and tissue-specific enzyme development in ascidian embryos. Blastomeres were dissociated from 44∽64-ceIl Halocynthia roretzi embryos and fractionated by centrifugation through a discontinuous Percoll density gradient. When cells harvested from the fraction at the bottom of the tube were division-arrested with cytochalasin B (an inhibitor of cytokinesis) soon after their isolation, more than 70% of them developed histochemically-detectable muscle-specific acetylcholinesterase (AChE) activity, suggesting that they were almost all blastomeres of muscle lineage. When these cells were arrested with aphidicolin (an inhibitor of DNA replication) and cytochalasin B immediately after their isolation, however, none of them showed AChE activity. When they were allowed to divide once and then arrested with the inhibitors, nearly 40 % of them developed AChE activity, and when they were allowed to divide twice before arrest, about 70% of them showed AChE activity.

Cholinergic enzyme activity in neurons of the developing anuran spinal cord.

Cholinergic enzyme activity was investigated over the course of spinal cord development from early larval (tadpole) stages to adult life in bullfrogs (Rana catesbeiana). Acetylcholinesterase (AChE) activity examined histochemically in spinal neurons and AChE and choline acetyltransferase (ChAT) activities were measured biochemically in axons of developing hindlimb motoneurons. At early larval stages, only spinal neurons born during embryonic life (primary neurons) showed histochemical evidence of AChE activity. Hindlimb motoneuron somata did not show AChE activity until mid-larval stages. AChE and ChAT activities were found in hindlimb motoneuron axons at the earliest stages examined, when the hindlimb consists of a small bud of undifferentiated mesenchyme. Activities of both enzymes show steady increases over the course of development until climax, when activities maintain a plateau until metamorphosis is complete. Total activities of both enzymes increase as the adult frog grows, although ChAT activity shows a much greater proportional increase than AChE activity.

The vegetative network in the guinea pig and rat sacral spinal cords.

The intermediate zones in the sacral segments of guinea pig and rat spinal cords are histochemically investigated for the presence of catecholaminergic and cholinergic structures. The presence of a well-developed noradrenergic network is demonstrated. This network connects the preganglionic vegetative nuclei in horizontal and vertical directions. In addition, only some fibers from the bundles composing this network show acetylcholinesterase activity.

Autonomic nervous system of the cat middle ear mucosa.

The autonomic nerves of the cat middle ear mucosa were studied by the methods of surface preparation, histochemistry and electron microscopy. The tympanic nerve was found to contain large myelinated fibers probably of a sensory nature. Other sympathetic bundles consisted primarily of unmyelinated fibers with a sparse distribution of myelinated fibers. The inferior tympanic artery was accompanied by many vasomotor nerves and its branches were also innervated predominantly by adrenergic fibers. A dense neural network which showed acetylcholinesterase activity was found in the mucosa around the Eustachian tube orifice. These nerves consisted of several unmyelinated fibers encapsulated by a single Schwann cell just below the epithelium. The fine structure of the small ganglions of the tympanic plexus revealed that the cholinergic nerve endings synapse with the cell body of the ganglion. This finding suggests that these ganglions are autonomic.

Innervation of human axillary sweat glands. Histochemical and electron microscopic study of hyperhidrotic and normal subjects.

Adrenergic nerves were demonstrated by formaldehyde-induced fluorescence and cholinergic nerves using thiocholine techniques to demonstrate acetylcholinesterase activities at the light and electron microscopic levels. The specimens were taken during surgery from the axillae of hyperhidrotic patients and normal voluntary controls. No fluorescent nerves were found around eccrine or apocrine sweat glands in hyperhidrotic or normally sweating axillae. Both eccrine and apocrine sweat glands exhibited a nerve network showing acetylcholinesterase activity. There was no marked difference in the innervation patterns or in the intensity of the acetylcholinesterase reaction of the nerves in the hyperhidrotic patients or normal subjects. Ultrastructurally the acetylcholinesterase-positive nerves were seen in the vicinity of both eccrine and apocrine glands, but these nerves were outside the basement membrane.

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.

Differentiation of sympathicoblasts in cultures of chick ganglia: light and electron microscopic, fluorescence and enzyme histochemical observations.

Immature sympathetic ganglia prepared from 5 1/2-or 6-day-old chick embryos were cultured up to one month. The in vitro development was followed by phase microscopy, electron microscopy and using histochemistry for catecholamines, monoamine oxidase and cholinesterases. During the first week of culture extensive plexuses of nerve fibres were formed between and around the clusters of nerve cells. Mature-looking neurons were observed in the cultures by phase microscopy after three weeks, at which age the mean diameter of the perikarya was more than doubled. Varying catecholamine fluorescence was observed in the perikarya during the entire culture period. The nerve fibres showed usually only weak fluorescence, but, in the older cultures, bright varicosities were regularly found in the fibres. Monoamine oxidase activity was demonstrated already at three days of culture and the reaction was maintained positive. Weak or moderate acetyl-cholinesterase activity was demonstrated in the sympathicoblasts and young sympathetic neurons and their processes. The axolemma showed acetylcholinesterase activity also around the nerve terminals containing small dense cored vesicles. Reactions for the non-specific cholinesterases were negative. Electron microscopy of the 30-day-old cultures revealed that the clusters of nerve cells consisted of mature sympathetic neurons, which contained large (60-200 nm) and small (35-60 nm) granular catecholamine-storing vesicles. Glial cells were almost totally lacking. Large numbers of nerve terminals containing both large and small granular vesicles were observed in the clusters, often in synaptic contact with the sympathetic neurons. It is concluded that the primitive sympathicoblasts are, in favourable conditions, capable of differentiation in culture up to mature sympathetic neurons.

The distribution of monoamine oxidase and acetylcholinesterase in the brain of Xenopus laevis tadpoles.

The distribution of monoamine oxidase (MAO) in the brain of Xenopus laevis tadpoles (stage 52–56) was studied histochemically with a modified Glenner's tryptamine-tetrazolium method. A moderate activity was observed in fibre regions of the striatum and septum (including the medial and lateral forebrain bundles), in the neuropil of the nucleus amygdalae, in the commissura anterior and commissura hippocampi, in the fibre regions of the diencephalon (including the optic chiasma), in the fibre regions of the tectum opticum and the tegmentum of the mesencephalon and in the white substance of the ventral half of the medulla oblongata. A greater MAO activity was found in the neuropil of the entire nucleus praeopticus. In the partes anterior and magnocellularis of this nucleus, MAO positive fibres are present in close contact with the perikarya, indicating a monoaminergic innervation of these neurons. The perikarya themselves did not show MAO activity. In the neurons of the nucleus praeopticus epichiasmaticus, the paraventricular organ (PVO) and nucleus infundibularis dorsalis (NID), only a slight MAO activity has been demonstrated in the perikarya, whereas a strong MAO positivity was found in the intraventricular protrusions and the neuropil. These data indicate the aminergic character of the neurons of these nuclei. From the postoptic fibre region a MAO positive tract was observed towards the developing median eminence and pars intermedia of the hypophysis. The pars nervosa and some cells of the pars distalis also contained MAO. Along the border of the aquaeduct of Silvius and the fourth ventricle, MAO positive liquor-containing neurons are also present. The distribution of acetylcholinesterase (AChE) was investigated in the hypothalamohypophysial region. AChE activity was found in the neuropil of the nucleus praeopticus magnocellularis, in the fibres of the optic chiasma and in the postoptic fibre region. The neurons of the PVO and NID were AChE negative. An AChE positive tract could be traced from the postoptic fibre region to the developing median eminence and pars nervosa. The pars distalis did not show AChE activity. However, in tadpoles reaching the metamorphic climax, ChE activity appeared in certain cells of the pars distalis; this might be related to degenerative phenomena in the acidophilic cells. The absence of AChE activity in the pars intermedia indicates a regulation of MSH release by peptidergic nerves to be unlikely.

Catecholamines and cholinesterases in the paracervical (Frankenhäuser) ganglion of normal and pregnant rats.

AbstractChatecholamines and cholinesterases were studied histochemically in the uterine paracervical ganglion of normal and pregnant rats and after both sympathectomy and sacral parasympathectomy. About one fifth of the ganglion cells revealed intense acetylcholinesterase (AChE) activity. The cells were large and devoid of catecholamines and received AChE‐containing nerves. — About one third of the ganglion cells contained weak to moderate yellow‐green catecholamine fluorescence. They were presumed to be adrenergic although they also exhibited moderate to weak AChE activity. These postganglionic neurons represent “short” adrenergic neurons. The rest of the ganglion cells with moderate to weak AChE activity were presumed to be cholinergic. Increased ganglion cell fluorescence was observed after 2 weeks of pregnancy. There was no change in the cholinesterase activity of the neurons. — Division of the sympathetic or the sacral parasympathetic nerves did not cause changes in the intensity of the histochemical reactions. The small, intensely fluorescent cells (SIF‐cells) did not show AChE activity. Other cholinesterases, although present in the cytoplasm of the ganglion cells and SIF‐cells, were mainly localized in the glial cells.

The lingual taste buds of Gallus domesticus L.

Synopsis The so‐called “ taste buds “ of the chicken were examined. It was found that they had one or more pores and were composed of a single cell type. There was a nerve plexus at the base of the bud giving rise to fibres which entered the bud. The cells showed acetyl cholinesterase activity and were found to degenerate following denervation. Thus they closely parallel, in both structure and neural dependency, the taste buds in other vertebrates and it is concluded that they probably are true taste buds.

Histochemical study of the acetylcholinesterase activity in the inner ear of the squirrel monkey.

Perfusion-fixation was performed on the squirrel monkey, whose temporal bones were then decalcified in cold EDTA (ethylenediamine tetracetate) solution. Gholinesterase (ChE) activity was demonstrated on frozen sections of these bones. ChE in the efferent fibers of the inner ear was identified as acetylcholinesterase (AChE) by using specific inhibitors. Fine, scattered AChE containing fibers in the cochlear nerve were observed crossing the glial-Schwann sheath junction which were not a part of Rasmussen's bundle. After transection of the cochlear nerve, these fine fibers lost AChE activity, suggesting they were of efferent nature. In the organ of Corti, both the upper tunnel radial and basilar bundles were found to show AChE activity. These two bundles apparently contained efferent fibers.