Caenorhabditis elegans: localization of cholinesterase associated with anterior nematode structures.

By employing a histochemical procedure on adult nematodes, the base of the Caenorhabditis elegans amphid appears to contain acetylcholinesterase and a nonspecific cholinesterase. Some precipitation was observed in the kinetosome region of the inner labial papilla with acethylthiocholine (AtCh) as substrate but not, in limited observations, in the absence of substrate or with butyrylthiocholine (BtCh). The amphidial tips, the tips of the inner labial papillae, and the lining of the buccal cavity contained substantial reaction product at the ultrastructural level, with or without substrates and inhibitors and therefore cannot be related to the presence of a cholinesterase.     

Peripheral ChE Inhibition Modulates Brain Monoamines Levels and <Emphasis Type="Italic">c-fos</Emphasis> Oncogene in Mice Subjected to a Stress Situation

The present study examined, in mice, whether regional patterns of brain monoamines concentrations (DA, 5-HT and their metabolites) and expression of c-Fos protein, that may represent a prolonged functional change in neurons, could be changed after a combined exposure to stress and the peripheral cholinesterase reversible inhibitor pyridostigmine (PYR). Animals were subjected every day to a random combination of mild unescapable electric footshocks and immobilization over a 12-day period, resulting in a significant increase of glucocorticoids levels and an activation of c-fos in hippocampus, thalamus and piriform cortex. This stress protocol induced a significant increase of 5-HT levels in striatum, hippocampus and ponto mesencephalic area (PMA) but failed to induce any DA activation. When PYR (0.2 mg/kg s.c. inducing 19–35% inhibition of the plasmatic ChE activity) was administered twice a day during the last 5 days of the stress session, 5-HIAA levels and expression of c-fos oncogene were significantly increased in the most of the brain areas studied. DA levels were also enhanced in striatum/hippocampus as a result of a possible activation of mesolimbic and nigrostriatal dopamine systems. Taken together, these results suggest that a combined exposure to certain stress conditions and PYR leads, in mice, to functional changes in neurons and may affect centrally controlled functions. The mechanisms underlying these modifications and their behavioral implications remain to be further investigated.     

Localization of Butyrylcholinesterase at the Neuromuscular Junction of Normal and Acetylcholinesterase Knockout Mice

At the mouse neuromuscular junction (NMJ), there are two distinct cholinesterases (ChE): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Until now, it has been difficult to determine the precise localization of BChE at the NMJ. In this study, we use a modification of Koelle''s method to stain AChE and BChE activity. This method does not interfere with fluorescent co-staining, which allows precise co-localization of ChE and other synaptic molecules at the NMJ. We demonstrate that AChE and BChE exhibit different localization patterns at the mouse NMJ. AChE activity is present both in the primary cleft and in the secondary folds, whereas BChE activity appears to be almost absent in the primary cleft and to be concentrated in subsynaptic folds. The same localization for BChE is observed in the AChE-knockout (KO) mouse NMJ. Collagenase treatment removed AChE from the primary cleft, but not from secondary folds in the wild-type mouse, whereas in the AChE-KO mouse, BChE remains in the secondary folds. After peripheral nerve injury and regeneration, BChE localization is not modified in either normal or KO mice. In conclusion, specific localization of BChE in the secondary folds of the NMJ suggests that this enzyme is not a strict surrogate of AChE and that the two enzymes have two different roles. (J Histochem Cytochem 58:1075–1082, 2010)     

Study of cholinesterases of different origin by the method of inhibitor analysis (variation of structure of phosphoryl and leaving parts of dialkoxyphosphates)

Analytical review is performed of literature data on inhibitor analysis of structure of cholinesterases from different animals (vertebrates, insects, molluscs) by studying rate of their interaction with 190 dialkoxyphosphates of 54 homologous series with regularly changed structure of their phosphoryl group. The presented data are discussed from the point of view of comparative biochemistry and in the light of current concepts of structure of cholinesterase active center in animals at different levels of evolutionary development.     

How thionphosphonates inhibit activity of different cholinesterases

Analysis of mechanism of reversible inhibition of human erythrocyte acetylcholinesterase (AChE), of horse serum cholinesterase (ChE), and ChE of optical ganglia tissue of individuals of the Commander squid Berryleuthis magister from various habitat zones was studied under effect of thionphosphonates (P=S), derivatives of piperidine, morpholine, perhydroazepine as well as several heterocyclic model compounds. Data of comparative inhibitory specificity have allowed us to suggest that thionphosphonates are sorbed in the area of cholinesterase esterase center through the phosphoryl part of the inhibitor molecule, rather than through its heterocyclic grouping. An advantage in the antienzyme efficiency of thionphosphonates (P=S) over phosphonates (P=O) is revealed. Effect of the ion strength is used for analysis of contribution of the hydrophobic—hydrophilic interaction in the enzyme—inhibitor system.     

Carboxylic ester hydrolases: Classification and database derived from their primary,secondary, and tertiary structures

We classified the carboxylic ester hydrolases (CEHs) into families and clans by use of multiple sequence alignments, secondary structure analysis, and tertiary structure superpositions. Our work for the first time has fully established their systematic structural classification. Family members have similar primary, secondary, and tertiary structures, and their active sites and reaction mechanisms are conserved. Families may be gathered into clans by their having similar secondary and tertiary structures, even though primary structures of members of different families are not similar. CEHs were gathered from public databases by use of Basic Local Alignment Search Tool (BLAST) and divided into 91 families, with 36 families being grouped into five clans. Members of one clan have standard α/β‐hydrolase folds, while those of other two clans have similar folds but with different sequences of their β‐strands. The other two clans have members with six‐bladed β‐propeller and three‐α‐helix bundle tertiary structures. Those families not in clans have a large variety of structures or have no members with known structures. At the time of writing, the 91 families contained 321,830 primary structures and 1378 tertiary structures. From these data, we constructed an accessible database: CASTLE (CArboxylic eSTer hydroLasEs, http://www.castle.cbe.iastate.edu ).     

Structural and functional investigations of cholinesterases by means of affinity electrophoresis

1. After a brief survey of the basic affinity electrophoresis concepts, the usual ways for preparing affinity electrophoresis ligands are examined. 2. Then results obtained on cholinesterases are reviewed. This section includes (a) structural and functional investigations on anionic sites, i.e., study of ligand-induced conformational change, organophosphate-induced "aging," genetic variants, and active-site topology; and (b) characterization of cholinesterase conjugates (hybrid proteins) and glycoinositol phospholipid-anchored cholinesterases. 3. The future prospects of affinity electrophoresis, e.g., investigations on the esteratic site and exploration of the carbohydrate moiety, are emphasized in the concluding section.     

Human intestine epithelial cell acetyl- and butyrylcholinesterase

The epithelial cells of the human intestine exhibit a cholinesterase activity which is restricted to the apex of the villi. This activity displays a maximum in the colon and a minimum in the jejunum. Contrary to most of the studied vertebrates, the human cells present both acetylcholinesterase and butyrylcholinesterase activities, acetylcholinesterase being predominant in all the intestinal segments: duodenum, jejunum, ileum and colon. Like in the other vertebrates, only globular forms are identified by sucrose gradient centrifugation. However, the simultaneous presence, on the one hand of three globular forms (G₁, G₂ and G₄) and, on the other hand of soluble as well as detergent-soluble molecular species seems to be a particular feature of the human cells.Abbreviations ChE Cholinesterases - AChE Acetylcholinesterase - BuChE Butyrylcholinesterase     

Development of pyrazole and spiropyrazoline analogs as multifunctional agents for treatment of Alzheimer’s disease

Cholinergic hypothesis of Alzheimer’s disease has been advocated as an essential tool in the last couple of decades for the drug development. Here in, we report de novo fragment growing strategy for the design of novel 3,5-diarylpyrazoles and hit optimization of spiropyrazoline derivatives as acetyl cholinesterase inhibitors. Both type of scaffolds numbering forty compounds were synthesized and evaluated for their potencies against AChE, BuChE and PAMPA. Introduction of lipophilic cyclohexane ring in 3,5-diarylpyrazole analogs led to spiropyrazoline derivatives, which facilitated and improved the potencies. Compound 44 (AChE = 1.937 ± 0.066 µM; BuChE = 1.166 ± 0.088 µM; hAChE = 1.758 ± 0.095 µM; P_e = 9.491 ± 0.34 × 10⁻⁶ cm s¹) showed positive results, which on further optimization led to the development of compound 67 (AChE = 0.464 ± 0.166 µM; BuChE = 0.754 ± 0.121 µM; hAChE = 0.472 ± 0.042 µM; P_e = 13.92 ± 0.022 × 10⁻⁶ cm s¹). Compounds 44 and 67 produced significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE. They were found to be safer to MC65 cells and decreased metal induced Aβ_1-42 aggregation. Further, in-vivo behavioral studies, on scopolamine induced amnesia model, the compounds resulted in better percentage spontaneous alternation scores and were safe, had no influence on locomotion in tested animal groups at dose of 3 mg/kg. Early pharmacokinetic assessment of optimized hit molecules was supportive for further drug development.     

Purification and characterisation of a carboxylesterase from the latex ofSynadenium grantii Hook, ‘f’

The latex ofSynadenium grantii was found to contain esterolytic activity. Polyacrylamide gel electrophoretic study coupled with substrate and inhibitor specificity studies revealed the presence of multiple forms of carboxylesterases and cholinesterases in the latex. One of the carboxylesterases of the latex was purified by acetone fractionation, carboxymethyl-Sephadex chromatography and Sepharose-6B gel filtration. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme consists of a single polypeptide chain with a molecular weight of 14,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to basic amino acid residues. The isoelectric pH of the enzyme was found to be 4.0. The enzyme was a glycoprotein as revealed by periodic acid Schiff-staining technique. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was sensitive to organophosphates. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol.