• Korean journal of applied entomology
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• v.31 no.2
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• pp.122-132
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• 1992

Experiments were conducted to establish an in vitro AChE inhibition test system to evaluate the potency of AChE inhibition of new chemical compounds. For a fixed time inhibition test, optimal inhibition (incubation) time to evaluate their AChE inhibition potency was 10 min. for AChE inhibitors such as DFP, DDVP, and paraoxon. The concentration of new chemical compounds with an ester group for evaluation of their inhibition potency was 10 $\mu$M under 10 min. preincubation conditions. However, the stepwise inhibition test with higher concentrations seemed to be needed for other chemical compounds. For a progressive inhibition test to calculate inhibition constants such as $K_d$, $K_3$ and $K_i$, extremely low $K_d(1.3\times10-^85.6\times10^{-7})$ and $K_3$(0. 21-0.27 $min^{-1}$) were observed under lagged preincubation time (0.8-13.3 min) and low in¬hibitor concentrations $(1\times10-^92\times10-^6M)$. However, this method seemed to be useful for comparison of AChE inhibition potency among inhibitors. Differences in inhibition potency among DFP, paraoxon, and KH501 were due to the differences in $K_d$, in other words, differences in affinities between inhibitors and AChEs. Therefore, AntiChE screening should consist of two steps. The first step is to evaluate the potency of AChE inhibition based on $I_50$ valuse obtained from fixed time inhibition tests. The second step is to study inhibition patterns and characteristics of chemical compounds selected in the first step.

• Journal of Environmental Science International
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• v.17 no.12
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• pp.1325-1330
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• 2008

This study was carried out with the detection for multiresidue of the carbamate pesticide such as carbaryl and cabofuran by enzyme-inhibition method. The check time for determination of acetylcholinesterase(AChE) activity was selected at 60 sec. The AChE activity in chicken brain determined by the Ellman's method was $162{\mu}$mol/min/g protein. $I_{50}$ for AChE by carbamate pesticide with wet kit was 0.169mg/L of carbaryl and 0.089mg/L of cabofuran, respectively. The incubation time for enzyme kit with substrate kit was 30min for determination of AChE activity. Enzyme kit with substrate kit was stable at $4^{\circ}C\;and\;25^{\circ}C$ for 5 days. Limit detection concentration of carbaryl with dry kit for AChE was 0.05mg/L. The dry kit such as wet kit applied Enzyme-Inhibition(EI) method with AChE was confirmed the multi residue method to detect the carbamate pesticides.

• Environmental Analysis Health and Toxicology
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• v.17 no.3
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• pp.265-272
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• 2002

This study was carried out with the detection for multiresidue of the organophosphorus pesticides such as malathion, parathion. diazinon, and carbamate pesticide such as carbaryl, by enzyme-inhibition method. The acetylcholinesterase (AChE) and cholinesterase (ChE) activities in chicken brain determined by the Ellman's method were 166.6 and 5.8 $\mu$mol/min/g protein, and in chicken plasma were 23.1 and 8.3 $\mu$mol/min/g protein, respectively. The optimum pH of AChE and ChE was 8.2 and 7.8, respectively. The Km of AChE and ChE was 0.034 and 0.045 mM, respectively. I$\_$50/ for AChE and ChE by some organophosphorus was 55.82 and 99.42 mg/L of malathion, 31.16 and 29.13 mg/L of parathion, and 17.89 and 19.62 mg/L of diazinon, respectively. I$\_$50/ for AChE and ChE by carbaryl of carbamate was 0.10 and 0.05 mg/L, respectively. The 0.07 mg/L of drinking water advisory level for carbaryl could be detected with I$\_$50/ of AChE and ChE. Enzyme-Inhibition (EI) method with AChE and ChE was used the multiresidue method to detect the 1 mg/L of the carbamate pesticides.

• Archives of Pharmacal Research
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• v.14 no.4
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• pp.330-335
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• 1991

The effects of physostigmine and neostigmine on the parathin induced toxicity were examined in adult female rats. Physostigmine $(100\;{\mu}g/kg,\;ip)$ or neostigmine $(200\;{\mu}g/kg,\;ip)$ inhibited acetylcholinesterase (AChE) and cholinesterase (ChE) activities in blood, brain and lung when the enzyme activity was measured 30 min after the treatment. At the doses of two carbamates equipotent on brain AChE, neostigmine showed greater inhibition on peripheral AChE/ChE. The enzyme activity returned to normal in 120 min following the carbamates except in the lung of rats treated with neostigmine. Carbamates administered 30 min prior to parathion (2 mg/kg) antagonized the inhibition of AChE/ChE by parathion when the enzyme activity was measured 2 hr following parathion. Neostigmine showed greater protective effect on peripheral AChE/ChE. The effect of either carbamate on AChE/ChE was not significant 2 hr beyond the parathion treatment. Carbamates decreased the mortality of rats challenged with a lethal dose of parathion (4 mg/kg, ip) either when treated alone or in combination with atropine (10 mg/kg, ip). Lethal action of paraoxon (1.5 mg/ks ip), the active metabolite of parathion was also decreased by the carbamate treatment indicating that the protection was not mediated by competitive inhibition of metabolic conversion of parathion to paraoxon. The results suggest that carbamylation of the active sites may not be the sole underlying mechanism of protection provided by the carbamates.

• Food Engineering Progress
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• v.15 no.4
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• pp.289-296
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• 2011

Twenty four fractions by solvent extraction and/or acid precipitation from fruit body and culture broth of Inonotus obliquus were prepared, and their inhibitory effect against acetylcholinesterase (AChE) was investigated. Among these fractions, acid (1 M HCl) precipitates from cell-free culture broth and fruit body exhibited the highest inhibitory effect on AChE in vitro. Acid precipitates inhibited AChE activity in a concentration-dependant manner and $IC_{50}$ values of both acid precipitates were 0.53 mg/mL. The inhibition pattern was general non-competitive inhibition. The energetic parameters were also determined by dual substrate/temperature design. Both acid precipitates increased the values of Ea, ${\Delta}H,/;{\Delta}G$ and ${\Delta}H^{\ast}$ decreasing the value of ${\Delta}S$ for AChE. The results implied that the acid precipitates from I. obliquus increased the thermodynamic barrier, leading to the breakdown of ES complex and the formation of products as inhibitory mechanism.

• Applied Biological Chemistry
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• v.38 no.2
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• pp.174-178
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• 1995

The purpose of this study was to investigate a role of cytochrome $P_{450}$, for the toxicity of the phosalone in in vitro and in vivo bioactivation systems. The bimolecular inhibition rate constants$(k_i)$ of the phosalone to acetylcholinesterase(AChE) and butyrylcholinesterase(BuChE) were approximately $10^2M^{-1}{\cdot}min^{-1}$, respectively, which meant a poor inhibitor. The potency of the phosalone as an inhibitor of AChE and BuChE was increased about 300 and 40 fold, respectively, when the inhibitor and the ChE were incubated with microsomes fortified with NADPH compared with microsome alone. Piperonyl butoxide(PB) addition to these coupled systems greatly reduced the inhibition of both target enzymes by blocking a bioactivation process. The $I_{50}$ value of the Phosalone alone for rat brain AChE was 170 mg/kg. When PB was pretreated, that value was altered to 42.5 mg/kg. PB pretreatment synergized the inhibition of brain AChE with four times. Rat blood erythrocyte AChE and plasma BuChE were similarly inhibited in vivo by the phosalone and PB pretreatment didn't affect significantly the pattern of the inhibition. The in vivo studies showed different results in the role of cytochrome $P_{450}$ from those of the in vitro studies.

• The Korean Journal of Pesticide Science
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• v.2 no.2
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• pp.10-15
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• 1998

The bimolecular inhibition rate constants of carbofuran and N-dimethylphosphinothioyl carbofuran(PSC) to acetylcholinesterase(AChE) were $7.7{\times}10^{5}\;M^{-1}{\cdot}min^{-1}$ and $1.2{\times}10^{3}\;M^{-1}{\cdot}min^{-1}$, respectively. These results showed that PSC required a bioactivation process for its toxic action because it didn't inhibit the target enzyme effectively. The potency of PSC as an inhibitor of AChE increased when PSC and AChE were incubated with microsomes fortified with NADPH compared with microsome alone. Piperonyl butoxide(PBO) addition to these coupled systems greatly reduced the inhibition of the target enzyme by blocking the bioactivation process. In vivo inhibition study of mouse brain AChE, $I_{50}$ value for AChE was 28 mg/kg for PSC and the value increased to 57 mg/kg when PBO was pretreated. This result showed that cytochrome $P_{450}$ would also play a role in the bioactivation process of PSC in vivo. And conversioin of carbofuran from PSC was 55 % in a chemical oxidation system using meta-chloroperoxybenzoic acid. The oxidative activation of PSC to carbofuran was shown to be essential for showing its toxicological action and cytochrome $P_{450}$ was identified as an important enzyme which participated in this process.

• Korean journal of applied entomology
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• v.31 no.1
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• pp.1-6
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• 1992

These studies were carried out to compare toxicological test methods of two spotted spider mite, Tetranychus urticae Koch,; and to investigate relationship between in vivo resistant level of highly acaricide-selected population, and in vitro insensitivity of the AChE in the same population to carbophenothion and ethion. The slide dip method (CV = 8.7%) was of more accuracy and suitability than that of the leaf dip method(CV=12.2%) and leaf disc method (CV= 13.6 %) in determination of the resistant levels of twospotted spider mite to acaricides. The slide dip method also had the advantages of simple treatment with different populations on a slide at the same time, standardization of post-treatement conditions and living plants exclud¬ed from the test. Even though the topical application method(CV =8.1 %) showed high accuracy, it had the demerits of the much time consuming, need of expensive equipment and difficulty of test manipulation. For a limited time, the 22nd successive carbophenothion-selected population of two-spotted spider mite showed 156- and 128-fold resistant levels to carbophenothion and ethion(both alPs), respectively. However, the 24th successive ethion-selected population revealed 64.1- and 65-fold resistant levels to ethion and carbophenothion, respectively. In the inhibition of AChE activity, the carbophenothion-selected population showed 3.3- and 2.7-fold insensitivity in AChE activity to carbophenothion and ethion, respectively. Likewise, the ethion -selected population exhibited 3- and 2.6-fold insensitivity in AChE activity to carbophenothion and ethion, respectively, as compared with that of susceptible population. As a result, a good relation was recognized between in vivo resistance to organophosphorous acaricides and in vitro insensitivity of the AChE to corresponding inhibitors.

• BMB Reports
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• v.43 no.8
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• pp.573-578
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• 2010

Several organophosphorus (OP) insecticides can selectively kill the silkworm maggot, Exorista sorbillans (Es) (Diptera: Tachinidae), while not obviously affecting the host (Bombyx mori) larvae, but the mechanism is not yet clear. In this study, the cDNA encoding an acetylcholinesterase (AChE) from the field Es was isolated. One point mutation (Gly353Ala) was identified. The Es-353G AChE and Es-353A AChE were expressed in baculovirus-insect cell system, respectively. The inhibition results showed that for eserine and Chlorpyrifos, Es-353A AChE was significantly less sensitive than Es-353G AChE. Meanwhile, comparison of the I50 values of eserine, dichlorvos, Chlorpyrifos and omethoate of recombinant Es AChEs with its host (Bombyx mori) AChEs indicated that, both Es AChEs are more sensitive than B. mori AChEs. The results give an insight of the mechanism that some OP insecticides can selectively kills Es while without distinct effect on its host, B. mori.

• Bulletin of the Korean Chemical Society
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• v.35 no.6
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• pp.1681-1686
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• 2014

A series of hybrid molecules between (${\alpha}$)-lipoic acid (ALA) and 4-amino-1-benzyl piperidines were synthesized and their in vitro cholinesterase (acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)) inhibitory activities were evaluated. Even though the parent compounds did not exhibit any inhibitory activity against cholinesterase (ChE) with the exception of compound 14 ($IC_{50}=255.26{\pm}4.41$ against BuChE), all hybrid molecules demonstrated BuChE inhibitory activity. Some hybrid compounds also displayed AChE inhibitory activity. Specifically, compound 17 was shown to be an effective inhibitor against both AChE ($IC_{50}=1.75{\pm}0.30{\mu}M$) and BuChE ($IC_{50}=5.61{\pm}1.25{\mu}M$) comparable to galantamine ($IC_{50}=1.7{\pm}0.9{\mu}M$ against AChE and $IC_{50}=9.4{\pm}2.5{\mu}M$ against BuChE). Inhibition kinetic studies using compound 17 indicated a mixed inhibition type for AChE and a noncompetitive inhibition type for BuChE. Its binding affinity ($K_i$) values to AChE and BuChE were $3.8{\pm}0.005{\mu}M$ and $7.0{\pm}0.04{\mu}M$, respectively.