Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase an...Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase（BChE, EC 3.1.1.8）, which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene（h BCh E） as published. The ratio of guanine and cytosine in four kinds of bases（（G＋C） ratio） was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg m L^–1 with an activity of 14.7 U m L^–1, which was purified 3.2×10^3-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg^–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range（〉60% activity at pH 4.0 to 8.0）. Moreover, it had a good adaptability to high temperature（〉60% activity at both 50 and 60°C up to 60 min） and good stability at 70°C. The enzyme can be activated by Li^＋, Co^＋, Zn^2＋ and ethylene diamine tetraacetic acid（EDTA）, but inhibited by Mg^2＋, Mn^2＋, Fe^2＋, Ag^＋ and Ca^2＋. Na^＋ had little effect on its activity. The values of h BChE of the Michaelis constant（Km） and maximum reaction velocity（Vm） were 89.4 mmol L^–1 and 1 721 mmol min^–1 mg^–1, respectively. The bim olecular rate constants（K_i） of the hBChE to four pesticides were similar with that of electric eel AChE（EeAChE） and higher than that of horse BChE（HoBChE）. All vlues of the half maximal inhibitory concentration of a substance（IC50） for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.展开更多
Developmental exposure to organophosphate insecticide is well known to induce neurobehavioral impairments, at late period. The present study aims to investigate the effects of subchronic exposure to Dimethoate, on loc...Developmental exposure to organophosphate insecticide is well known to induce neurobehavioral impairments, at late period. The present study aims to investigate the effects of subchronic exposure to Dimethoate, on locomotors skills and anxiety like behavior among wistar rat. Two groups of female’s rats are used. The intoxicated group receives daily, during five weeks, by intragastric gavage, a dose of Dimethoate dissolved in corn oil (100 mg/kg body weight). The control group receives only the corn oil. Spontaneous locomotors activity is evaluated using the Open Field test (OF) and anxiety-like behavior is measured using Elevated Plus-Maze (EPM). Dimethoate induced significant impairment of spontaneous locomotors activities, which is reflected by high decrease of number of squares crossed (SC) in OF. Females exposed to Dimethoate develop further anxiety-like response, expressed by significant reductions of the time spent in open arm of Elevated Plus-Maze.展开更多
Developmental exposure to organophosphate insecticide is well known to induce neurobeha-vioral impairments, at late period. The present study aims to investigate the effects of chronic exposure to Malathion, from in u...Developmental exposure to organophosphate insecticide is well known to induce neurobeha-vioral impairments, at late period. The present study aims to investigate the effects of chronic exposure to Malathion, from in utero to young adult stage, on locomotor skills and anxiety like- behavior among wistar rat. Four groups of female rats, bred with one non-pesticide exposed male, are used. On gestational day 6, three groups receive daily, by intragastric gavage, 3 different doses of Malathion dissolved in corn oil (100, 200 and 300 mg/kg body weight). The control group receives the corn oil only. On postnatal day 21, weaned offsprings are submitted to the similar treatment until adult age. Spontaneous locomotor activity is evaluated using the Open-Field test (OF) and anxiety-like behavior is measured using both Open-Field (OF) test and Elevate Plus-Maze (EPM). Malathion at 300 mg/kg is toxic to pregnant dams, and pups are stillborns. In males, Malathionlevelat 100 and 200 mg/kg induced significant impairment of spontaneous locomotor activities, which is reflected by high decrease of number of squares crossed in OF. In contrast, no discernible changes are observed within females Malathion-treated-group. However, females exposed to both malathion levels develop further anxiety-like response, expressed by significant reductions of exploratory activities in OF and time spent in open arm of EPM. Neurochemistry assay shows that cerebellum and neocortex acetylcholinesterase (AChE) activity inhibition are significantly increased with neurobehavioral deficits in males, relative to females. Overall, neurobehavioral outcomes of current study reveal that developmental exposure to Malathion induces sex-selective effects with greater changes in females.展开更多
基金financially supported by the Rural Work Committee of Beijing City for the First Batch of Agricultural Science and Technology Project of Beijing University of Agriculture, China (2013010102)the Beijing Innovation Team Building Project of Leafy Vegetables of Modern Agricultural Industry System, China (2063213003)the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions, China (CIT&TCD 20150315)
文摘Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase（BChE, EC 3.1.1.8）, which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene（h BCh E） as published. The ratio of guanine and cytosine in four kinds of bases（（G＋C） ratio） was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg m L^–1 with an activity of 14.7 U m L^–1, which was purified 3.2×10^3-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg^–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range（〉60% activity at pH 4.0 to 8.0）. Moreover, it had a good adaptability to high temperature（〉60% activity at both 50 and 60°C up to 60 min） and good stability at 70°C. The enzyme can be activated by Li^＋, Co^＋, Zn^2＋ and ethylene diamine tetraacetic acid（EDTA）, but inhibited by Mg^2＋, Mn^2＋, Fe^2＋, Ag^＋ and Ca^2＋. Na^＋ had little effect on its activity. The values of h BChE of the Michaelis constant（Km） and maximum reaction velocity（Vm） were 89.4 mmol L^–1 and 1 721 mmol min^–1 mg^–1, respectively. The bim olecular rate constants（K_i） of the hBChE to four pesticides were similar with that of electric eel AChE（EeAChE） and higher than that of horse BChE（HoBChE）. All vlues of the half maximal inhibitory concentration of a substance（IC50） for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.
文摘Developmental exposure to organophosphate insecticide is well known to induce neurobehavioral impairments, at late period. The present study aims to investigate the effects of subchronic exposure to Dimethoate, on locomotors skills and anxiety like behavior among wistar rat. Two groups of female’s rats are used. The intoxicated group receives daily, during five weeks, by intragastric gavage, a dose of Dimethoate dissolved in corn oil (100 mg/kg body weight). The control group receives only the corn oil. Spontaneous locomotors activity is evaluated using the Open Field test (OF) and anxiety-like behavior is measured using Elevated Plus-Maze (EPM). Dimethoate induced significant impairment of spontaneous locomotors activities, which is reflected by high decrease of number of squares crossed (SC) in OF. Females exposed to Dimethoate develop further anxiety-like response, expressed by significant reductions of the time spent in open arm of Elevated Plus-Maze.
文摘Developmental exposure to organophosphate insecticide is well known to induce neurobeha-vioral impairments, at late period. The present study aims to investigate the effects of chronic exposure to Malathion, from in utero to young adult stage, on locomotor skills and anxiety like- behavior among wistar rat. Four groups of female rats, bred with one non-pesticide exposed male, are used. On gestational day 6, three groups receive daily, by intragastric gavage, 3 different doses of Malathion dissolved in corn oil (100, 200 and 300 mg/kg body weight). The control group receives the corn oil only. On postnatal day 21, weaned offsprings are submitted to the similar treatment until adult age. Spontaneous locomotor activity is evaluated using the Open-Field test (OF) and anxiety-like behavior is measured using both Open-Field (OF) test and Elevate Plus-Maze (EPM). Malathion at 300 mg/kg is toxic to pregnant dams, and pups are stillborns. In males, Malathionlevelat 100 and 200 mg/kg induced significant impairment of spontaneous locomotor activities, which is reflected by high decrease of number of squares crossed in OF. In contrast, no discernible changes are observed within females Malathion-treated-group. However, females exposed to both malathion levels develop further anxiety-like response, expressed by significant reductions of exploratory activities in OF and time spent in open arm of EPM. Neurochemistry assay shows that cerebellum and neocortex acetylcholinesterase (AChE) activity inhibition are significantly increased with neurobehavioral deficits in males, relative to females. Overall, neurobehavioral outcomes of current study reveal that developmental exposure to Malathion induces sex-selective effects with greater changes in females.
