The Experts below are selected from a list of 117 Experts worldwide ranked by ideXlab platform
John G. Hildebrand - One of the best experts on this subject based on the ideXlab platform.
-
Distribution of acetylcholinesterase activity in the deutocerebrum of the sphinx moth Manduca sexta
Cell and Tissue Research, 1995Co-Authors: Uwe Homberg, Sally G. Hoskins, John G. HildebrandAbstract:We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta . To distinguish between extra-and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with Echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show Echothiophateinsensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, Echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with malespecific pheromone-sensitive afferents in the macro-glomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echo-thiophate-insensitive AChE activity. These neurous seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antenno-cerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In Echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine.
Oksana Lockridge - One of the best experts on this subject based on the ideXlab platform.
-
Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad.
Chemico-biological interactions, 2019Co-Authors: Bella L. Grigorenko, Dana A. Novichkova, Sofya V. Lushchekina, Irina V. Zueva, Lawrence M. Schopfer, Alexander V. Nemukhin, Sergey D. Varfolomeev, Oksana Lockridge, Patrick MassonAbstract:Abstract A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with Echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by Echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.
-
gene delivered butyrylcholinesterase is prophylactic against the toxicity of chemical warfare nerve agents and organophosphorus compounds
Journal of Pharmacology and Experimental Therapeutics, 2011Co-Authors: Kalpana Parikh, Oksana Lockridge, Ellen G. Duysen, Benjamin Snow, Neil S Jensen, Veeraswamy Manne, Nageswararao ChilukuriAbstract:Gene delivery using an adenoviral system has been effective in introducing therapeutic proteins in vitro and in vivo. This study tested the feasibility of using adenovirus to deliver clinically relevant amounts of butyrylcholinesterase (BChE), a proven bioscavenger of nerve agents. The adenovirus construct expressed full-length mouse BChE. Mice were injected with a single dose of adenovirus (1.5 × 10(10) infectious units) in the tail vein; plasma was collected through day 11 and assayed for BChE activity. Maximum activity, representing a 300- to 3400-fold increase over baseline, was found on day 4. Expression levels returned to baseline by day 10. Nondenaturing gel electrophoresis showed the recombinant BChE was a dimer that could be converted to tetramers by addition of polyproline. The toxic compounds chosen for protection studies were positively charged organophosphorus agents, Echothiophate, and O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX). Mice containing elevated blood levels of BChE (300- to 3,000-fold over the control mice) were challenged with incremental doses of Echothiophate or VX. Mice showed no signs of toxicity and were protected from up to 30× LD(50) dose of Echothiophate and 5× LD(50) dose of VX. A good correlation was observed between tolerated Echothiophate dose and plasma BChE levels at time of challenge. The absolute increases in levels of circulating BChE and the sustained nature of the response resulted in a very high enzyme concentration, deemed critical in acute toxicity (5× LD(50) or more) scenarios. These results suggest that gene-delivered BChE is a prophylactic and affords protection equivalent to that of a multimilligram injection of the same.
-
Aging Pathways for Organophosphate-Inhibited Human Butyrylcholinesterase, Including Novel Pathways for Isomalathion, Resolved by Mass Spectrometry
2007Co-Authors: Lawrence M. Schopfer, Patrick Masson, Marie Therese Froment, Florian Nachon, Oksana LockridgeAbstract:Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser–O–P(O)–(Y)–(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X–R moiety is dealkylated by breaking either the P–X or the X–R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, Echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O 18 to allow incorporation of 18 O, if cleavage was at the P–X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25°C, pH 7.0. However, BChE inhibited by dichlorvos, Echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O–C bond cleavage, i.e., the classical X–R scission pathway. In contrast, isomalathion aged through both X–R and P–X pathways; the main aged product resulted from P–S bond cleavage and a minor product resulted from O–C and/or S–C bond cleavage. Key Words: butyrylcholinesterase; organophosphate; aging; mass spectrometry
-
Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants
Analytical biochemistry, 2004Co-Authors: Yuxia Wang, Lawrence M. Schopfer, Patrick Masson, Ellen G. Duysen, Florian Nachon, Oksana LockridgeAbstract:Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM Echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM Echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with Echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.
-
Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase.
Toxicology and applied pharmacology, 2004Co-Authors: Yuxia Wang, Andreea Ticu Boeck, Ellen G. Duysen, Margaret L. Van Keuren, Thomas L. Saunders, Oksana LockridgeAbstract:Organophosphorus toxicants (OP) include chemical nerve agents and pesticides. The goal of this work was to find out whether an animal could be made resistant to OP toxicity by genetic engineering. The human butyrylcholinesterase (BChE) mutant G117H was chosen for study because it has the unusual ability to hydrolyze OP as well as acetylcholine, and it is resistant to inhibition by OP. Human G117H BChE, under the control of the ROSA26 promoter, was expressed in all tissues of transgenic mice. A stable transgenic mouse line expressed 0.5 microg/ml of human G117H BChE in plasma as well as 2 microg/ml of wild-type mouse BChE. Intestine, kidneys, stomach, lungs, heart, spleen, liver, brain, and muscle expressed 0.6-0.15 microg/g of G117H BChE. Transgenic mice were normal in behavior and fertility. The LD50 dose of Echothiophate for wild-type mice was 0.1 mg/kg sc. This dose caused severe cholinergic signs of toxicity and lethality in wild-type mice, but caused no deaths and only mild toxicity in transgenic animals. The mechanism of protection was investigated by measuring acetylcholinesterase (AChE) and BChE activity. It was found that AChE and endogenous BChE were inhibited to the same extent in Echothiophate-treated wild type and transgenic mice. This led to the hypothesis that protection against Echothiophate toxicity was not explained by hydrolysis of Echothiophate. In conclusion, the transgenic G117H BChE mouse demonstrates the factors required to achieve protection from OP toxicity in a vertebrate animal.
Richard P. Winne - One of the best experts on this subject based on the ideXlab platform.
-
Intrathecal acetyl cholinesterase inhibitors produce analgesia that is synergistic with morphine and clonidine in rats.
Anesthesia and analgesia, 1995Co-Authors: Stephen E. Abram, Richard P. WinneAbstract:Intrathecal (IT) administration of acetyl cholinesterase inhibitors produces analgesia through a muscarinic action.The addition of IT cholinergic agonists to IT opioids or alpha2-adrenergic agonists results in enhanced analgesic effects, but it is not clear whether these interactions are synergistic, additive, or less than additive. Dose-response curves for hot plate and tail immersion tests were established for IT neostigmine, physostigmine, and Echothiophate in rats. Dose-response curves for hot plate testing were established for IT morphine and clonidine. The effect of maximally effective doses of each of the three cholinergic drugs on hot plate testing was plotted over time. Isobolographic analysis was performed for hot plate testing using neostigmine-morphine and neostigmine-clonidine combinations. The three cholinesterase inhibitors produced profound analgesia on hot plate testing but incomplete analgesia using the tail immersion test. Duration of analgesia on hot plate testing ranged from 45 min for physostigmine to more than 24 h for Echothiophate. IT administration of combinations of neostigmine plus morphine and neostigmine plus clonidine both produced significantly more profound analgesia than the calculated additive effects and are, therefore, synergistic in their actions. (Anesth Analg 1995;81:501-7)
-
Intrathecal acetyl cholinesterase inhibitors produce analgesia that is synergistic with morphine and clonidine in rats.
Anesthesia and analgesia, 1995Co-Authors: Stephen E. Abram, Richard P. WinneAbstract:Intrathecal (IT) administration of acetyl cholinesterase inhibitors produces analgesia through a muscarinic action. The addition of IT cholinergic agonists to IT opioids or alpha 2-adrenergic agonists results in enhanced analgesic effects, but it is not clear whether these interactions are synergistic, additive, or less than additive. Dose-response curves for hot plate and tail immersion tests were established for IT neostigmine, physostigmine, and Echothiophate in rats. Dose-response curves for hot plate testing were established for IT morphine and clonidine. The effect of maximally effective doses of each of the three cholinergic drugs on hot plate testing was plotted over time. Isobolographic analysis was performed for hot plate testing using neostigmine-morphine and neostigmine-clonidine combinations. The three cholinesterase inhibitors produced profound analgesia on hot plate testing but incomplete analgesia using the tail immersion test. Duration of analgesia on hot plate testing ranged from 45 min for physostigmine to more than 24 h for Echothiophate. IT administration of combinations of neostigmine plus morphine and neostigmine plus clonidine both produced significantly more profound analgesia than the calculated additive effects and are, therefore, synergistic in their actions.
Uwe Homberg - One of the best experts on this subject based on the ideXlab platform.
-
Distribution of acetylcholinesterase activity in the deutocerebrum of the sphinx moth Manduca sexta
Cell and Tissue Research, 1995Co-Authors: Uwe Homberg, Sally G. Hoskins, John G. HildebrandAbstract:We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta . To distinguish between extra-and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with Echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show Echothiophateinsensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, Echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with malespecific pheromone-sensitive afferents in the macro-glomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echo-thiophate-insensitive AChE activity. These neurous seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antenno-cerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In Echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine.
Stephen E. Abram - One of the best experts on this subject based on the ideXlab platform.
-
Intrathecal acetyl cholinesterase inhibitors produce analgesia that is synergistic with morphine and clonidine in rats.
Anesthesia and analgesia, 1995Co-Authors: Stephen E. Abram, Richard P. WinneAbstract:Intrathecal (IT) administration of acetyl cholinesterase inhibitors produces analgesia through a muscarinic action.The addition of IT cholinergic agonists to IT opioids or alpha2-adrenergic agonists results in enhanced analgesic effects, but it is not clear whether these interactions are synergistic, additive, or less than additive. Dose-response curves for hot plate and tail immersion tests were established for IT neostigmine, physostigmine, and Echothiophate in rats. Dose-response curves for hot plate testing were established for IT morphine and clonidine. The effect of maximally effective doses of each of the three cholinergic drugs on hot plate testing was plotted over time. Isobolographic analysis was performed for hot plate testing using neostigmine-morphine and neostigmine-clonidine combinations. The three cholinesterase inhibitors produced profound analgesia on hot plate testing but incomplete analgesia using the tail immersion test. Duration of analgesia on hot plate testing ranged from 45 min for physostigmine to more than 24 h for Echothiophate. IT administration of combinations of neostigmine plus morphine and neostigmine plus clonidine both produced significantly more profound analgesia than the calculated additive effects and are, therefore, synergistic in their actions. (Anesth Analg 1995;81:501-7)
-
Intrathecal acetyl cholinesterase inhibitors produce analgesia that is synergistic with morphine and clonidine in rats.
Anesthesia and analgesia, 1995Co-Authors: Stephen E. Abram, Richard P. WinneAbstract:Intrathecal (IT) administration of acetyl cholinesterase inhibitors produces analgesia through a muscarinic action. The addition of IT cholinergic agonists to IT opioids or alpha 2-adrenergic agonists results in enhanced analgesic effects, but it is not clear whether these interactions are synergistic, additive, or less than additive. Dose-response curves for hot plate and tail immersion tests were established for IT neostigmine, physostigmine, and Echothiophate in rats. Dose-response curves for hot plate testing were established for IT morphine and clonidine. The effect of maximally effective doses of each of the three cholinergic drugs on hot plate testing was plotted over time. Isobolographic analysis was performed for hot plate testing using neostigmine-morphine and neostigmine-clonidine combinations. The three cholinesterase inhibitors produced profound analgesia on hot plate testing but incomplete analgesia using the tail immersion test. Duration of analgesia on hot plate testing ranged from 45 min for physostigmine to more than 24 h for Echothiophate. IT administration of combinations of neostigmine plus morphine and neostigmine plus clonidine both produced significantly more profound analgesia than the calculated additive effects and are, therefore, synergistic in their actions.
