Why are there so few resistance-associated mutations in insecticide target genes?
(1/2082)
The genes encoding the three major targets of conventional insecticides are: Rdl, which encodes a gamma-aminobutyric acid receptor subunit (RDL); para, which encodes a voltage-gated sodium channel (PARA); and Ace, which encodes insect acetylcholinesterase (AChE). Interestingly, despite the complexity of the encoded receptors or enzymes, very few amino acid residues are replaced in different resistant insects: one within RDL, two within PARA and three or more within AChE. Here we examine the possible reasons underlying this extreme conservation by looking at the aspects of receptor and/or enzyme function that may constrain replacements to such a limited number of residues. (+info)
Calcitonin gene-related peptide decreases expression of acetylcholinesterase in mammalian myotubes.
(2/2082)
Nerve-derived trophic factors are known to modulate expression of acetylcholinesterase (AChE) in skeletal muscle fibers, yet the precise identity of these factors remains elusive. In the present study, we treated mouse C2 myotubes with calcitonin gene-related peptide (CGRP). Compared to non-treated myotubes, cell-associated AChE activity levels were decreased by approximately 60% after 48 h of treatment. A parallel reduction in AChE total protein levels was also observed as determined by Western blot analysis. The reduction in AChE activity was due to a decrease in the levels of the G1 molecular form and to an elimination of G1. By contrast, levels of secreted AChE remained unchanged following CGRP treatment. Finally, the overall decrease in AChE activity was accompanied by a reduction in AChE transcripts which could not be attributed to changes in the transcriptional rate of the ACHE gene. (+info)
Monoclonal antibody 3F3 against conformational epitope of Torpedo acetylcholinesterase.
(3/2082)
AIM: To study the type of epitope of native Torpedo acetylcholinesterase (AChE) directed by its monoclonal antibody (McAb) 3F3. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used for the assay of the reaction between antigen and antibody. RESULTS: McAb 3F3 immunoreacted well with the native AChE, but not with the reduced- and alkylated-AChE (RA-AChE) at all. Soman did not interfere the binding of 3F3 with AChE molecule. The synthesized 24-peptide containing the active serine residue of the AChE active center did not react with McAb 3F3. CONCLUSION: 3F3 is a monoclonal antibody against the conformational epitope of Torpedo AChE active center, but dose not occupy the active serine residue of the enzyme. (+info)
Establishment and characterization of human neuroblastoma cell lines.
(4/2082)
Three new tissue culture cell lines, CHP-100, CHP-126, and CHP-134, have been established from explant cultures of human neuroblastoma. The cell lines have been characterized with respect to morphology, chromosomes constitution, growth, neural enzyme content, and their ability to grow in nude mice. The cells grow as dense masses comprised of fibroblast-or neuroblast-like cells with small processes. The cell lines differ in their neural enzyme acitivity. The chromosomal content of the 3 cell lines is near diploid, and all are capable of forming tumors in nude mice. The morphological findings indicate that the cells in culture resemble those found in the tumor, and the enzyme activities are consistent with those of nervous tissue. This the morphological, biochemical, and tumorigenic properties confirm that the 3 cell lines are neoplastic cells of neural origin. (+info)
Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site.
(5/2082)
Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6, 6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim. (+info)
Organophosphorylation of acetylcholinesterase in the presence of peripheral site ligands. Distinct effects of propidium and fasciculin.
(6/2082)
Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogenic organophosphates, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expression system. First-order rate constants kOP were 1,600 +/- 300 min-1 for DEPQ and 150 +/- 11 min-1 for EMPC, and second-order rate constants kOP/KOP were 193 +/- 13 microM-1 min-1 for DEPQ and 0.7-1.0 +/- 0.1 microM-1 min-1 for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased kOP/KOP by factors of 2-20 for these organophosphates. Such modest inhibitory effects are consistent with our recently proposed steric blockade model (Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216). Moreover, the binding of propidium resulted in a clear increase in kOP for EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values of kOP/KOP were decreased by factors of 10(3) to 10(5), and kOP was decreased by factors of 300-4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin. (+info)
Genetic analysis of collagen Q: roles in acetylcholinesterase and butyrylcholinesterase assembly and in synaptic structure and function.
(7/2082)
Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ-/- mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ-/- muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ-/- neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity. (+info)
Cloning, expression, and properties of a nonneuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.
(8/2082)
We have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis. The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes. The residues in the catalytic triad are conserved, as are the six cysteines which form the three intramolecular disulfide bonds. Three of the fourteen aromatic residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues, corresponding to Tyr-70 (Thr), Trp-279 (Asn), and Phe-288 (Met). High level expression was obtained via secretion from Pichia pastoris. The purified enzyme behaved as a monomeric hydrophilic species. Although of invertebrate origin and possessing the above substitutions in the active site gorge residues, the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine. It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2. 5 mM and was highly sensitive to both active site and "peripheral" site inhibitors. Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection. (+info)