Ultrastructural demonstration of acetylcholinesterase activity of motor endplates via osmiophilic diazothioethers

Summary The ultrastructural chemical localization of acetylcholinesterase of motor endplates of rat intercostal muscle has been studied with three new esterase substrates. These substrates, although not specific for acetylcholinesterase, have differential affinities for various types of esterases; two of them (NTA and TAB) are hydrolyzed preferentially by acetyl esterase enzymes, and the third (TPB) is a propionic acid ester and is hydrolyzed preferentially by pseudocholinesterase and other esterases. The end-product of the enzymatic reaction is converted to a diazothioether (droplet form) and upon osmication this is converted to a coordination polymer of osmium which has ideal properties for electron microscopy.Although this study supports previous observations that enzymatic activity can be found primarily on the post-synaptic membranes of the motor endplate, no enzymatic activity was noted on the pre-synaptic membrane, within the synaptic cleft, or on the basement membrane unless incubation was prolonged, resulting in overstaining. Neither was enzyme activity seen on membrane-free ribosomes and the ribosome-studded sarcoplasmic reticulum. Axonal vesicles also failed to exhibit enzymatic activity which had been noted with the method using thiolacetic acid and lead. A correlation of esterase activity with ultrastructural localization, using the substrate TPB, suggests that a buffer zone of nonspecific esterase activity is present beneath the subneural apparatus which limits the aberrant, accidental, or abnormal distribution of acetylcholine within a clearly defined area of sarcoplasm in the vicinity of the motor endplate of the muscle fiber.This investigation was supported by research grants from the National Cancer Institute (CA-2078 and CA-02478) and National Institute of Neurological Diseases and Blindness (NB 04096). Acknowledgement for technical assistence is due Miss Julia Silhan.This investigation was carried out during the tenure of a Public Health Service research career program award NB 5820 from the N.I.N.D.B.     

Localization of acetylcholinesterase in the rat myoneural junction

Summary The distribution of myoneural acetylcholinesterase (AChE; EC. 3.1.1.7) was studied electron microscopically with the copper ferricyanide method at pH 6.0, using acetylthiocholine iodide as substrate and iso-OMPA to exclude other cholinesterase activity.It was observed that the results obtained with this method are affected by changes in the reaction temperature, inhibitor concentration and fixation time. Changes which retard the rate of hydrolysis of AChE were observed to transfer the localization of the reaction endproduct in intact junctions from the extracellular side of the postsynaptic membrane to the intracellular side of the postsynaptic membrane. From the results it is concluded that the site of most intense AChE corresponds to the region of increased subsarcolemmal electron density of the postsynaptic membrane.     

Localization of neuropeptide Y in human sympathetic ganglia: Correlation with met-enkephalin, tyrosine hydroxylase and acetylcholinesterase

Summary The relationships of immunoreactive neuropeptide Y, enkephalin and tyrosine hydroxylase, on the one hand, and acetylcholinesterase histochemical activity, on the other, were studied in human lumbar sympathetic ganglia. Two thirds of the ganglion cells contained immunoreactive neuropeptide Y. Electron microscopically the immunoreaction was localized in the Golgi apparatus and in large dense-cored vesicles in the nerve endings. Most of the neuropeptide-containing neurons and nerve fibres were also reactive for tyrosine hydroxylase. Nerve fibres reactive for neuropeptide Y were found around ganglion cells regardless of their transmitter contents, whereas enkephalin-reactive nerve terminals surrounded only acetylcholinesterase-containing neurons. The results demonstrate that neuropeptide Y is colocalized with noradrenaline in most of the human sympathetic neurons and that the nerve fibres may innervate selectively the noradrenergic and cholinergic subpopulations of ganglion cells depending on the transmitters of the nerves.     

From protein to peptides: a spectrum of non-hydrolytic functions of acetylcholinesterase

Acetylcholinesterase (AChE), a member of the α/β-hydrolase fold superfamily of proteins, is a serine hydrolase responsible for the hydrolysis of the well studied neurotransmitter acetylcholine (ACh). However, it is becoming clear that AChE has a range of actions other than this 'classical' role. Non-classical AChE functions have been identified in apoptosis, stress-responses, neuritogenesis, and neurodegeneration. Furthermore, these non-classical roles are attributable not only to the native protein, which appears to act as a mediary binding protein under a number of circumstances, but also to peptides cleaved from the parent protein. Peptides cleaved from AChE can act as independent signalling molecules. Here we discuss the implications of non-hydrolytic functions of this multi-tasking protein.     

Localization of emulsan-like polymers associated with the cell surface of acinetobacter calcoaceticus

Various immunochemical techniques were employed to probe the relationship between the extracellular emulsifying agent (emulsan) and the cell-associated form of the polymer in Acinetobacter calcoaceticus RAG-1. Using an emulsan-specific antibody preparation, immunocytochemical labeling revealed that an emulsan-like antigen is a major component of the 125-nm minicapsule which envelopes the exponential-phase cell of the parent strain. The marked reduction of this capsule in stationary-phase cells was correlated with the production of extracellular emulsifying activity. Crossed immunoelectrophoresis techniques demonstrated that the major antigenic component (S1) of the culture supernatant fluid is immunochemically identical to purified emulsan, yet electrophoretically distinct. The characteristics of the parent strain were compared with those of two phage-resistant mutant strains which are defective in extracellular emulsan production. One of these mutants, termed TR3, lacked both the emulsan-like capsule on the cell surface and the extracellular S1 component. A second phage-resistant emulsan-defective mutant (TL4) was characterized by an antigenically altered and inactive form of extracellular emulsan. A relatively small amount of emulsan-like capsular material was consistently demonstrated on the cell surface of this mutant. The correlation between phage sensitivity and extracellular emulsan production was strengthened by the fact that emulsan-specific antibodies inhibited both emulsification activity and phage adsortion onto cells of the parent strain.     

A new depsidone of Lobaria pulmonaria with acetylcholinesterase inhibition activity

The phytochemical investigation conducted on a foliose lichen, Lobaria pulmonaria(L.) Hoffm. (Lobariaceae), led to the isolation of a new depsidone in the form of its diacetate derivative which showed a moderate acetylcholinesterase inhibition activity (1 µg) in vitro. This is the first record of identified depsidone structure in searching for these inhibitors.     

Localization of acetylcholinesterase and choline acetyltransferase in the rat pituitary gland

Summary Experiments were conducted to determine the presence of two cholinergic biomarkers, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the rat pituitary. A histochemical procedure for AChE was used to provide visualization of structures containing this enzyme. Radiochemical methods provided a sensitive assay for measuring ChAT activity. Nerve fibres staining for AChE activity were observed in the neurointermediate lobe, with the greatest concentrations appearing at the junction region with the pituitary stalk. Cells staining for AChE were found in the pars distalis and pars intermedia. ChAT activity correlated well with AChE distribution in pars nervosa and pars intermedia but not in pars distalis. The greatest levels of ChAT activity were in pars intermedia and the region where the stalk joins the pituitary. Significant values were also found for the pars nervosa. The presence of AChE and ChAT in pars intermedia and pars nervosa is evidence for a cholinergic innervation to these regions. In pars distalis, where other investigators have found muscarinic receptors, intense staining for AChE and absence of ChAT activity may indicate non-innervated, acetylcholine-sensitive sites.     

Lipase-catalysed synthesis of new acetylcholinesterase inhibitors: N-benzylpiperidine aminoacid derivatives

New acetylcholinesterase inhibitors were synthetized via a lipase-mediated regioselective amidation using Candida antarctica lipase B as a biocatalyst in the key step. The new compounds have two different structural fragments: a N-benzylpiperidine moiety to anchor the enzyme active site and a dicarboxylic aminoacid to act as a biological carrier. Some analogues of N-benzylpiperazine were also synthesised and studied but they did not display AChE inhibitor activity. A preliminary structure activity relationship study was performed employing some computational techniques as similarity indices and electrostatic potential maps.     

Control of xyloglucan endotransglucosylase activity by salts and anionic polymers

Crude extracts of cauliflower florets had high xyloglucan endotransglucosylase (XET) activity, but this was largely lost after partial purification and de-salting. Activity was restored (promoted up to 40-fold) by any of a wide variety of inorganic and organic salts. Optimum concentrations for Na⁺, K⁺ and NH₄⁺ salts were typically ~300 mM. The chlorides of Ca²⁺, Mg²⁺, Al³⁺ and La³⁺ were optimally active at lower concentrations (e.g. 0.1 mM LaCl₃), but became inhibitory at higher concentrations (e.g. 5 mM LaCl₃). Some anionic polysaccharides at 0.04–0.2% w/v (e.g. gum arabic, pectin and hypochlorite-oxidised xyloglucan) promoted the XET activity of de-salted enzyme, especially if a sub-optimal concentration of NaCl was also present; others (e.g. homogalacturonan, 4-O-methyl-glucuronoxylan and alginate) were inhibitory. Similar ionic effects were noted on the XET activity of the Arabidopsis protein XTH24 (heterologously expressed by insect cells); in this case carboxymethylcellulose was also stimulatory. To look for endogenous modulators of XET activity, we prepared a cold-water extract of cauliflower florets; after boiling and centrifugation, the supernatant [boiled cauliflower preparation (BCP)] promoted the XET activity of de-salted cauliflower enzyme and of XTH24. About half the activator present in BCP was an ethanol-precipitable, anionic polymer of apparent M_r <5,000. After acid hydrolysis the polymer yielded much arabinose and galactose, and small amounts of galacturonic and glucuronic acids amino acids were also present. The polymer may thus contain arabinogalactan-proteins. We suggest that acidic polymers and/or other apoplastic ions are naturally occurring regulators of XET action in vivo, and may thus control cell wall assembly, loosening, and growth.Abbreviations AGP Arabinogalactan-protein - BCP Boiled cauliflower preparation (cold-water-extract of cauliflower florets that was then boiled) - CMC Carboxymethylcellulose - DE Degree of esterification - GalA Galacturonic acid - GlcA Glucuronic acid - K_av Elution volume relative to those of Blue Dextran (K_av=0) and glucose (K_av=1) - TFA Trifluoroacetic acid - V₀ Void volume (centre of elution peak of Blue Dextran) - V_i Totally included volume (centre of elution peak of glucose) - XEH Xyloglucan endohydrolase (activity) - XET Xyloglucan endotransglucosylase (activity) - XLLGol A xyloglucan-derived oligosaccharide, xylose₃·glucose₃·galactose₂·glucitol - XTH Xyloglucan endotransglucosylase/hydrolase (protein) - µ Ionic strength     

Potent acetylcholinesterase inhibitors: design, synthesis and structure-activity relationships of alkylene linked bis-galanthamine and galanthamine-galanthaminium salts

The syntheses, the anticholinesterase activities and structure-activity relationships of homodimeric (3a-c) and heterodimeric (6a-c) alkylene linked bis-galanthamine are reported. Compounds 6b-c were found to be more potent than galanthamine and tacrine in inhibiting AChE.     

Localization and possible functions of phospholipase D isozymes

The activation of PLD is believed to play an important role in the regulation of cell function and cell fate by extracellular signal molecules. Multiple PLD activities have been characterized in mammalian cells and, more recently, several PLD genes have been cloned. Current evidence indicates that diverse PLD activities are localized in most, if not all, cellular organelles, where they are likely to subserve different functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics.     

Tests with Daphnia magna: A new approach to prescreen toxicity of newly synthesized acetylcholinesterase reactivators

Reactivators of phosphorylated acetylcholinesterase (oximes) are substances used as a human antidotal therapy for organophosphate poisoning. The objective of our study was to examine if juveniles of freshwater microcrustacean Daphnia magna could be employed as test animals in early screen toxicity tests of those substances as a first step for further experiments with daphnids intoxicated by organophosphates. For this purpose, seven different oximes were investigated. It was found that toxicity of all tested oximes increased with time. Mono-quaternary oximes were approximately ten fold (EC₅₀, 14.9 mg.l^{? 1}) more toxic in 24 hour tests and five fold (EC₅₀ was 79.46 mg.l^{? 1}) more toxic in 48 hour tests than bis-quaternary oximes. Tests with daphnids were shown to be easy to carry out at low cost and provided valuable results which could be used as a starting point for further research.     

Tests with Daphnia magna: a new approach to prescreen toxicity of newly synthesized acetylcholinesterase reactivators

Reactivators of phosphorylated acetylcholinesterase (oximes) are substances used as a human antidotal therapy for organophosphate poisoning. The objective of our study was to examine if juveniles of freshwater microcrustacean Daphnia magna could be employed as test animals in early screen toxicity tests of those substances as a first step for further experiments with daphnids intoxicated by organophosphates. For this purpose, seven different oximes were investigated. It was found that toxicity of all tested oximes increased with time. Mono-quaternary oximes were approximately ten fold (EC50, 14.9 mg.l(-1)) more toxic in 24 hour tests and five fold (EC50 was 79.46 mg.l(-1)) more toxic in 48 hour tests than bis-quaternary oximes. Tests with daphnids were shown to be easy to carry out at low cost and provided valuable results which could be used as a starting point for further research.     

Development of new acetylcholinesterase reactivators: Molecular modeling versus in vitro data

In this work a theoretical methodology for evaluation of the association and kinetic reactivation constants of oximes using the Molegro^® and Spartan^® softwares was proposed and validated facing in vitro data previously reported in the literature. Results showed a very good agreement between the theoretical binding free energies of the reactivators and experimental data, suggesting that the proposed methodology could work well in the prediction of kinetic and thermodynamics parameters for oximes that might be helpful for the design and selection of new and more effective oximes.