[ Objective ] The paper was to systemically study the characteristics of acetyl cholinesterase （ACHE） in Pardosa astrigera, and confirm the occurrence and development of its pesticide resistance and the resistance l...[ Objective ] The paper was to systemically study the characteristics of acetyl cholinesterase （ACHE） in Pardosa astrigera, and confirm the occurrence and development of its pesticide resistance and the resistance level, thereby establishing a quick and accurate detection method for enzyme activity. [ Method] The optimal conditions for assaying the activity of AChE in different parts of P. astrigera were determined by orthogonal experiment. The distribution conditions of acetyl cholinesterase （AChE） in different tissues of P. astrigera were further studied, and the sensitivities of the enzyme tO four common pesticides were also determined. [ Result] The optimal condition for assaying the activity of AChE in cephalothorax, abdomen and appendage of P. astrigera was as follows, enzyme concentrations： 12, 18 and 29 g/L; substrate concentrations： 0.6, 1.0 and 1.0 mmol/L; pH value, 7.0; reaction temperatures： 30, 35, 35℃ ; reaction time, 5 rain. AChE was mainly distributed in the cephalothorax of P. astrigera. The specific activity of AChE in the extract solution with Triton X-100 was higher than that in the solution without Triton X-100. The median inhibitory concentrations （IC50） of methomyl, phoxim, betacypermethrin, chlorpyrifos against AChE in the cephalothorax of P. astrigera were 7.76 × 10^-5, 1.76×10^-4, 4.12 ×10^-4 and 4.94 ×10^-4 mol/L, respectively. [ Conclusion] AChEs in P. astrigera were membrane-bounded. The inhibition of four pesticides against AChE in the cephalothorax of P. astrigera had good dese-effect, this indicated that AChE in the cephalothorax of P. astrig- era could be used as the biochemical marker to monitor the contamination of organic phosphorus, carbamate and pyrethroid pesticides in the environment.展开更多
Acetylcholinesterase(AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method.An accidental discovery of a mutant strain with AChE activity was made in our laboratory du...Acetylcholinesterase(AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method.An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris.The pPIC9 K-Drosophila melanogaster acetylcholinesterase(DmAChE)-like expression vector was constructed by codon optimization of this mutant strain,which was transformed into P.pastoris GS115,and positive clones were selected on yeast peptone dextrose(YPD) plate with G418 at 4.0 mg/mL.The GS115-pPIC9 K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h,with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) pattern of the fermentation supernatant.After preliminary purification by ammonium sulfate precipitation,the enzyme activity was detected to be 76.9 U/(mL·min).In addition,the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.展开更多
目的:探究任督针刺结合经颅磁刺激(transcranial magnetic Stimulation,TMS)对血管性痴呆(vascular dementia,VD)患者血清中枢神经特异蛋白(central nervous system specific proteinβ,S100β)和乙酰胆碱酯酶(acetylcholinesterase,Ac...目的:探究任督针刺结合经颅磁刺激(transcranial magnetic Stimulation,TMS)对血管性痴呆(vascular dementia,VD)患者血清中枢神经特异蛋白(central nervous system specific proteinβ,S100β)和乙酰胆碱酯酶(acetylcholinesterase,AchE)的影响。方法:从广州市第十二人民医院收集2023年1月至2024年7月期间入院诊疗的VD患者90例,按照数字表法随机分为3组,即对照组、TMS组及针刺组,各30例。其中对照组给予口服药物治疗,TMS组基于对照组给予TMS治疗,针刺组则基于对照组、TMS组加用任督针刺治疗。持续治疗12周后,比较3组临床疗效;治疗前后血清S100β蛋白、血浆AchE水平及简易智力状态检查量表(mini-mental State Examination,MMSE)评分、日常生活活动能力(activities of daily living,ADL)评分。结果:针刺组、TMS组临床疗效有效率高于对照组(P<0.05)。治疗后3组血清S100β蛋白水平、MMSE评分及ADL评分较治疗前升高、血浆AchE水平较治疗前降低(P<0.05)。针刺组血清S100β蛋白水平、MMSE评分及ADL评分较TMS组、对照组高,血浆AchE水平较TMS组、对照组低(P<0.05);TMS组血清S100β蛋白水平、MMSE评分及ADL评分较对照组高,血浆AchE水平较对照组低(P<0.05)。结论:任督针刺结合TMS治疗VD患者的疗效甚佳,可调节血清S100β蛋白、血浆AchE水平,提升认知功能及日常活动能力。展开更多
To find promising new multitargeted AD （Alzheimer＇s disease） inhibitors, the 3D-QSAR （three-dimensional quantitative structure-activity relationship） model for 32 AD inhibitors was established by using the CoMFA ...To find promising new multitargeted AD （Alzheimer＇s disease） inhibitors, the 3D-QSAR （three-dimensional quantitative structure-activity relationship） model for 32 AD inhibitors was established by using the CoMFA （comparative molecular field analysis） and CoMSIA （comparative molecular similarity index analysis） methods. Results showed that the CoMFA and CoMSIA models were constructed successfully with a good cross-validated coefficient （q2） and a non-cross-validated coefficient （R2）, and the binding modes obtained by molecular docking were in agreement with the 3D-QSAR results, which suggests that the present 3D-QSAR model has good predictive capability to guide the design and structural modification of novel multitargeted AD inhibitors. Meanwhile, we found that one side of inhibitory molecule should be small group so that it would be conductive to enter the gorge to interact with the catalytic active sites of AChE （acetylcholinesterase）, and the other side of inhibitory molecule should be large group so that it would be favorable for interaction with the peripheral anionic site of ACHE. Furthermore, based on the 3D-QSAR model and the binding modes of AChE and [3-secretase （BACE-1）, the designed molecules could both act on dual binding sites of AChE （catalytic and peripheral sites） and dual targets （ACHE and BACE-1）. We hope that our results could provide hints for the design of new multitargeted AD derivatives with more potency and selective activity.展开更多
Acetylcholinesterase(AChE)plays a crucial role in the activities of the nervous system,and its abnormal function can lead to the occurrence and development of neurodegenerative diseases.Hence,an effective method for r...Acetylcholinesterase(AChE)plays a crucial role in the activities of the nervous system,and its abnormal function can lead to the occurrence and development of neurodegenerative diseases.Hence,an effective method for real-time monitoring of AChE activity is essential.Very recently,several fluorescence sensors have been developed for the detection of AChE activity,but they are usually imaging in the visible region,relatively small Stokes shifts,or long response times,limiting their application for real-time monitoring in vivo.Inspired by that,a near-infrared(NIR)off-on probe((E)-4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl dimethylcarbamate,DCM-N)for AChE monitoring with high selectivity and sensitivity is developed.In the probeDCM-N,a bright near-infrared fluorescence emission at 700 nmcan be triggered by AChE through the cleavage of amino ester bond in DCM-N,and the resulting fluorescence exhibits a good linear relationship with AChE activity in the range of 0.2e16 U/mL,with a detection limit as low as 0.06 U/mL.For real plasma sample detection,DCM-N demonstrates advantages of accurate detection and fast response compared to the traditional Ellman assay for AChE detection.Moreover,DCM-N can be used for imaging of AChE activity in live cells and tracking of AChE activity in zebrafish models,which is of great significance for medical and physiological research related to AChE.DCM-N possesses several notable features such as light-up NIR emission,fast response,large spectral shifts and strong photostability under physiological conditions.These features enable it to monitor AChE activity both in vivo and in vitro,providing a suitable tool for real-time monitoring and in vivo visualization of AChE activity.展开更多
Pyrrolizidine alkaloids(PAs)are natural toxins generated as secondarymetabolites in plants,predominantly consisting of unsaturated PAs with diverse toxicities,such as hepatotoxicity.Echimidine,a prominent PA,is believ...Pyrrolizidine alkaloids(PAs)are natural toxins generated as secondarymetabolites in plants,predominantly consisting of unsaturated PAs with diverse toxicities,such as hepatotoxicity.Echimidine,a prominent PA,is believed to exert various toxicological effects,including survival inhibition and induction of apoptosis of hepatocytes.However,the effects of echimidine on development remain unclear.We selected three concentrations of 0.02,0.2,and 2 mg/L to investigate the developmental toxicity of echimidine on zebrafish embryos.After a 7-day exposure,we observed hyperactivity and anxiety-like behavior in zebrafish larvae.Furthermore,we found that echimidine exposure significantly promoted embryonic motor neurodevelopment in geneticallymodified zebrafish.Next,we detected that echimidine exposure significantly increased the content of the excitatory neurotransmitter acetylcholine(ACh),accompanied by a significant decrease in acetylcholinesterase(AChE)activity.Conversely,echimidine led to a significant reduction in the content of the sedative neurotransmitterγ-aminobutyric acid(GABA),accompanied by abnormal gene expression of enzymes related to GABA synthesis.Moreover,we elucidated the strong direct binding of echimidine to zebrafish and human AChE protein through molecular docking.In summary,our study found that echimidine induced ACh accumulation possibly by inhibiting AChE activity,leading to motor neurodevelopmental abnormalities and hyperactivity in zebrafish larvae.This work provides important scientific knowledge on the effects and mechanisms of PAs on neural development,which is helpful for controlling the risk of PAs in food and protecting public health.展开更多
基金Supported by Shanxi Scholarship Fund(2009042)Youth Science andTechnology Research Fund in Shanxi Province(2008021041)~~
文摘[ Objective ] The paper was to systemically study the characteristics of acetyl cholinesterase （ACHE） in Pardosa astrigera, and confirm the occurrence and development of its pesticide resistance and the resistance level, thereby establishing a quick and accurate detection method for enzyme activity. [ Method] The optimal conditions for assaying the activity of AChE in different parts of P. astrigera were determined by orthogonal experiment. The distribution conditions of acetyl cholinesterase （AChE） in different tissues of P. astrigera were further studied, and the sensitivities of the enzyme tO four common pesticides were also determined. [ Result] The optimal condition for assaying the activity of AChE in cephalothorax, abdomen and appendage of P. astrigera was as follows, enzyme concentrations： 12, 18 and 29 g/L; substrate concentrations： 0.6, 1.0 and 1.0 mmol/L; pH value, 7.0; reaction temperatures： 30, 35, 35℃ ; reaction time, 5 rain. AChE was mainly distributed in the cephalothorax of P. astrigera. The specific activity of AChE in the extract solution with Triton X-100 was higher than that in the solution without Triton X-100. The median inhibitory concentrations （IC50） of methomyl, phoxim, betacypermethrin, chlorpyrifos against AChE in the cephalothorax of P. astrigera were 7.76 × 10^-5, 1.76×10^-4, 4.12 ×10^-4 and 4.94 ×10^-4 mol/L, respectively. [ Conclusion] AChEs in P. astrigera were membrane-bounded. The inhibition of four pesticides against AChE in the cephalothorax of P. astrigera had good dese-effect, this indicated that AChE in the cephalothorax of P. astrig- era could be used as the biochemical marker to monitor the contamination of organic phosphorus, carbamate and pyrethroid pesticides in the environment.
基金supported by the National Development and Reform Commission of China(No.20111158)。
文摘Acetylcholinesterase(AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method.An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris.The pPIC9 K-Drosophila melanogaster acetylcholinesterase(DmAChE)-like expression vector was constructed by codon optimization of this mutant strain,which was transformed into P.pastoris GS115,and positive clones were selected on yeast peptone dextrose(YPD) plate with G418 at 4.0 mg/mL.The GS115-pPIC9 K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h,with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) pattern of the fermentation supernatant.After preliminary purification by ammonium sulfate precipitation,the enzyme activity was detected to be 76.9 U/(mL·min).In addition,the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.
文摘目的:探究任督针刺结合经颅磁刺激(transcranial magnetic Stimulation,TMS)对血管性痴呆(vascular dementia,VD)患者血清中枢神经特异蛋白(central nervous system specific proteinβ,S100β)和乙酰胆碱酯酶(acetylcholinesterase,AchE)的影响。方法:从广州市第十二人民医院收集2023年1月至2024年7月期间入院诊疗的VD患者90例,按照数字表法随机分为3组,即对照组、TMS组及针刺组,各30例。其中对照组给予口服药物治疗,TMS组基于对照组给予TMS治疗,针刺组则基于对照组、TMS组加用任督针刺治疗。持续治疗12周后,比较3组临床疗效;治疗前后血清S100β蛋白、血浆AchE水平及简易智力状态检查量表(mini-mental State Examination,MMSE)评分、日常生活活动能力(activities of daily living,ADL)评分。结果:针刺组、TMS组临床疗效有效率高于对照组(P<0.05)。治疗后3组血清S100β蛋白水平、MMSE评分及ADL评分较治疗前升高、血浆AchE水平较治疗前降低(P<0.05)。针刺组血清S100β蛋白水平、MMSE评分及ADL评分较TMS组、对照组高,血浆AchE水平较TMS组、对照组低(P<0.05);TMS组血清S100β蛋白水平、MMSE评分及ADL评分较对照组高,血浆AchE水平较对照组低(P<0.05)。结论:任督针刺结合TMS治疗VD患者的疗效甚佳,可调节血清S100β蛋白、血浆AchE水平,提升认知功能及日常活动能力。
基金Acknowledgments The authors acknowledge the financial support of the Natural Science Foundation of Guangxi Province （No. 2013GXNSFAA019019） and the Natural Science Foundation of Guangxi Province （No. 2013GXNSFAA019041）.
文摘To find promising new multitargeted AD （Alzheimer＇s disease） inhibitors, the 3D-QSAR （three-dimensional quantitative structure-activity relationship） model for 32 AD inhibitors was established by using the CoMFA （comparative molecular field analysis） and CoMSIA （comparative molecular similarity index analysis） methods. Results showed that the CoMFA and CoMSIA models were constructed successfully with a good cross-validated coefficient （q2） and a non-cross-validated coefficient （R2）, and the binding modes obtained by molecular docking were in agreement with the 3D-QSAR results, which suggests that the present 3D-QSAR model has good predictive capability to guide the design and structural modification of novel multitargeted AD inhibitors. Meanwhile, we found that one side of inhibitory molecule should be small group so that it would be conductive to enter the gorge to interact with the catalytic active sites of AChE （acetylcholinesterase）, and the other side of inhibitory molecule should be large group so that it would be favorable for interaction with the peripheral anionic site of ACHE. Furthermore, based on the 3D-QSAR model and the binding modes of AChE and [3-secretase （BACE-1）, the designed molecules could both act on dual binding sites of AChE （catalytic and peripheral sites） and dual targets （ACHE and BACE-1）. We hope that our results could provide hints for the design of new multitargeted AD derivatives with more potency and selective activity.
基金supported by Jiangsu Innovation Team Program,China,National Natural Science Foundation of China(Grant Nos.:82204339,82473884,82122066,and 81973291)National Key Research and Development Program of Ministry of China(Grant No.:2022YFC2704603)+1 种基金the“Dawn”program of Shanghai Education Commission,China(Grant No.:22SG34)Natural Science Foundation of Sichuan Province of China(Grant No.:2023NSFSC1902).
文摘Acetylcholinesterase(AChE)plays a crucial role in the activities of the nervous system,and its abnormal function can lead to the occurrence and development of neurodegenerative diseases.Hence,an effective method for real-time monitoring of AChE activity is essential.Very recently,several fluorescence sensors have been developed for the detection of AChE activity,but they are usually imaging in the visible region,relatively small Stokes shifts,or long response times,limiting their application for real-time monitoring in vivo.Inspired by that,a near-infrared(NIR)off-on probe((E)-4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl dimethylcarbamate,DCM-N)for AChE monitoring with high selectivity and sensitivity is developed.In the probeDCM-N,a bright near-infrared fluorescence emission at 700 nmcan be triggered by AChE through the cleavage of amino ester bond in DCM-N,and the resulting fluorescence exhibits a good linear relationship with AChE activity in the range of 0.2e16 U/mL,with a detection limit as low as 0.06 U/mL.For real plasma sample detection,DCM-N demonstrates advantages of accurate detection and fast response compared to the traditional Ellman assay for AChE detection.Moreover,DCM-N can be used for imaging of AChE activity in live cells and tracking of AChE activity in zebrafish models,which is of great significance for medical and physiological research related to AChE.DCM-N possesses several notable features such as light-up NIR emission,fast response,large spectral shifts and strong photostability under physiological conditions.These features enable it to monitor AChE activity both in vivo and in vitro,providing a suitable tool for real-time monitoring and in vivo visualization of AChE activity.
基金supported by the Key Project at the Central Government Level:the ability establishment of sustainable use for valuable Chinese medicine resources(No.2060302)the National Natural Science Foundation of China(No.82104389)+2 种基金the Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences(No.CI2023E002)the High-level Key Discipline Program of National Administration of Traditional Chinese Medicine(No.ZYYZDXK-2023244)China Agricultural Research System of MOF and MARA(No.CARS-21).
文摘Pyrrolizidine alkaloids(PAs)are natural toxins generated as secondarymetabolites in plants,predominantly consisting of unsaturated PAs with diverse toxicities,such as hepatotoxicity.Echimidine,a prominent PA,is believed to exert various toxicological effects,including survival inhibition and induction of apoptosis of hepatocytes.However,the effects of echimidine on development remain unclear.We selected three concentrations of 0.02,0.2,and 2 mg/L to investigate the developmental toxicity of echimidine on zebrafish embryos.After a 7-day exposure,we observed hyperactivity and anxiety-like behavior in zebrafish larvae.Furthermore,we found that echimidine exposure significantly promoted embryonic motor neurodevelopment in geneticallymodified zebrafish.Next,we detected that echimidine exposure significantly increased the content of the excitatory neurotransmitter acetylcholine(ACh),accompanied by a significant decrease in acetylcholinesterase(AChE)activity.Conversely,echimidine led to a significant reduction in the content of the sedative neurotransmitterγ-aminobutyric acid(GABA),accompanied by abnormal gene expression of enzymes related to GABA synthesis.Moreover,we elucidated the strong direct binding of echimidine to zebrafish and human AChE protein through molecular docking.In summary,our study found that echimidine induced ACh accumulation possibly by inhibiting AChE activity,leading to motor neurodevelopmental abnormalities and hyperactivity in zebrafish larvae.This work provides important scientific knowledge on the effects and mechanisms of PAs on neural development,which is helpful for controlling the risk of PAs in food and protecting public health.
