氟化钠对小鼠全血乙酰明碱酯酶影响的动态观察

氟化钠对小鼠全血乙酰明碱酯酶影响的动态观察吴南屏，赵忠良，康富敏，韩宝红氟中毒患者有明显的乙酰胆碱蓄积症状，表明氟可抑制乙酰胆碱酯酶（ＡＣｈＥ）的活性，但文献报道染氟早期对ＡＣｈＥ活性影响结果不一［１－３］。本实验拟通过动态观察氟对小鼠全血ＡｃｈＥ活...

IL-1β对大鼠胆碱能神经元的影响

目的 探讨 IL- 1β对大鼠胆碱能神经元的损伤作用及机制。方法 将 IL- 1β缓慢释放颗粒 (IL- 1βpellet)植入大鼠右侧 Meynert核 (nb M,基底核 ) ,复制胆碱能系统受损的动物模型 ;将不同剂量的人重组白细胞介素 - 1β(rh IL- 1β)加入培养大鼠前脑基底神经元中 ,复制 rh IL- 1β诱导神经元损伤模型 ,检测 (乙酰胆碱酯酶 ) Ach E活性和脂质过氧化物 (L PO)含量的变化。结果 IL- 1β损伤侧皮质 Ach E活性较未损伤侧、正常组及假手术组均显著降低 (P <0 .0 1)。 5 0 - 5 0 0 U/ ml rh IL- 1β可引起培养的神经元 Ach E活性明显下降 (P <0 .0 5 ) ;10 0 - 5 0 0 U/ ml rh IL-1β可引起神经元 L PO含量明显升高 (P <0 .0 1)。结论 IL- 1β慢性作用可以造成大鼠中枢胆碱能系统的损害 。

Effect of Transgenic Bt plus CpTI Cotton on Carboxylesterase and Acetylcholinesterase of Cotton Aphid Aphis gossypii

[Objective] The research aimed to assess the effect of transgenic Bt plus CpTI cotton variety SGK321 on carboxylesterase and acetylcholinesterase of cotton aphid Aphis gossypii and provide theoretical basis for studying the biosafety of transgenic cotton.[Method] Cotton aphids were fed with SGK321 and Shiyuan321（normal parental varieties） for over 40 generations.Enzyme activities were compared between cotton aphids feeding on SGK321 for 1,2,3,41,42 and 43 generations with those on Shiyuan321.[Result] The carboxylesterase activity of cotton aphids feeding on SGK321 for 1 generation was significantly higher than those feeding on Shiyuan321.Acetylcholinesterase activity of cotton aphids feeding on SGK321 for 1,2 and 3 generations were significantly higher than those feeding on Shiyuan321 in the same generation.But there was no significant difference of enzyme activity between cotton aphids feeding on SGK321 for a long term and those feeding on parental cotton.[Conclusion] The cotton aphid that feeding on transgenic Bt plus CpTI cotton SGK321 for a long time has adaptivity to SGK321 by regulating the detoxifying enzyme.

Cloning and Sequencing of Two Acetylcholinesterase cDNA Fragments from Cotton Aphid,Aphis gossypii Glover

Two acetylcholinesterase (AChE) genes cDNA fragments,Ag.acel and Ag.ace2,have been cloned from cotton aphid,Aphis gossypii Glover using degenerate primers with RT-PCR technique.Ag.acel gene cDNA fragment is of 282?bp encoding 94 amino acids,and Ag.ace2 gene cDNA fragment is of 264?bp encoding 88 amino acids.Both two putative AChE genes cDNA fragments share numerous similarities with those cloned from other insects.This is the first report of two AChE cDNA fragment sequences in the insect species,which provided the direct evidence of multiple AChE existence in insects.

Morphological changes of cholinergic nerve fibers in the urinary bladder after establishment of artificial somatic-autonomic reflex arc in rats

Objective To establish an artificial somatic-autonomic reflex arc in rats and observe the following distributive changes of neural fibers in the bladder. Methods Adult Sprague-Dawley rats were randomly divided into three groups： control group, spinal cord injury （SCI） group, and reinnervation group. DiI retrograde tracing was used to verify establishment of the model and to investigate the transport function of the regenerated efferent axons in the new reflex arc. Choline acetyltransferase （CHAT） in the DiI-labeled neurons was detected by immunohistochemistry. Distribution of neural fibers in the bladder was observed by acetylcholine esterase staining. Results DR-labeled neurons distributed mainly in the left ventral horn from L3 to L5, and some of them were also CHAT-positive. The neural fibers in the bladder detrusor reduced remarkably in the SCI group compared with the control （P 〈 0.05）. After establishment of the somatic-autonomic reflex arc in the reinnervation group, the number of ipsilateral fibers in the bladder increased markedly compared with the SCI group （P 〈 0.05）, though still much less than that in the control （P 〈 0.05）. Conclusion The efferent branches of the somatic nerves may grow and replace the parasympathetic preganglionic axons through axonal regeneration. Acetylcholine is still the major neurotransmitter of the new reflex arc. The controllability of detrusor may be promoted when it is reinnervated by the pelvic ganglia efferent somatic motor fibers from the postganglionic axons.

Hirschsprung's disease:Is there a relationship between mast cells and nerve fibers?

AIM:To define the topography of mast cells and their numbers in cases of Hirschsprung's disease(HD)and non-HD,assess neural hypertrophy using imaging software and to study the relationship between mast cells and nerve fibers.METHODS:HE stained sections of 32 cases of chronic constipation in the age group of 0-14 years were reviewed for ganglion cells.AChE staining was performed on frozen sections of colonic and rectal biopsies.Based on their findings cases were divided into HD and non-HD and mast cells stained by toluidine blue were evaluated.Image analysis by computerized software was applied to S-100 stained sections for assessment of neural hypertrophy.RESULTS:Difference between number of mast cells in HD group(mean=36.44)and in non-HD group(mean =14.79)was statistically significant.Image analysis morphometry on S-100 stained sections served as a useful adjunct.The difference between number,size,and perimeter of the nerve fibers between HD and non-HD group was statistically significant.CONCLUSION:Mast cells are significantly increased in HD and their base line values are much higher in Indian children than that reported in Western literature.Their role in HD needs further research.Morphometry of S-100 stained nerve fibers is a useful adjunct to conventional methods for diagnosis of HD.

Development of a Zebrafish Model for Rapid Drug Screening against Alzheimer＇s Disease

Alzheimer＇s disease, the leading cause of dementia in the elderly, is a complex neurodegenerative disorder which leads to a progressive decline in cognitive functions. A rapid screening model is highly demanded for identification and evaluation of novel anti-Alzheimer＇s disease drugs from a large numbers of compounds. Until now, numerous studies utilized zebrafish model for drug discovery. Since aluminum can induce a similar biological activity in zebrafish as in Alzheimer patients, in this study, we developed a novel animal model using 3 to 5 day post-fertilization larval zebrafish by optimizing the doses and duration of aluminum chloride exposure. Six anti-Alzheimer＇s disease drugs with a variety of mechanisms were tested to validate the newly developed zebrafish model. Importantly, Rivastigmine, ThT, Flurbiprofen and AM-117 could increase the value of Dyskinesia Recovery Rate by 53.4-64%, 169.4-200%, 54.5-96% and 70.9-121%, respectively. Rivastigmine, Memantine, ThT, Flurbiprofen, Rosiglitazone and AM-117 improved the value of Response Efficiency by 86.6-175.1%, 28.2-66.6%, 127.2-236.5%, 118.3-323.7%, 26.6-140.8% and 70.2-161.4%, respectively. Our results suggest that the zebrafish model developed in this study could be a useful tool for high throughput screening of potential novel anti-Alzheimer＇s disease leading compounds targeting acetylcholinesterase, N-methyl-D-aspartic acid receptor, γ-secretase, peroxisome proliferator-activated receptor-γand amyloid-β.

Acute toxicity of organochlorine insecticide endosulfan to the giant freshwater prawn Macrobrochium rosenbergii

Endosulfan, an organochlorine pesticide, is highly toxic and effective at controlling pests in agriculture, horticulture, and public health programs. In this study, static bioassays were used to evaluate the toxicity of endosulfan to freshwater prawns( Macrobrachium rosenbergii) of various lengths(1.5±0.03,4±0.08, and 7±0.06 cm). Additionally, the activities of peroxidase(POD), acid phosphatase(ACP),alkaline phosphatase, acetylcholinesterase(AChE), and Na + /K +-ATPase were analyzed to refl ect the effects of endosulfan exposure. The 96 h LC 50 of endosulfan for prawns 1.5, 4, and 7 cm long were 1.86, 4.53,and 6.09μg/L, respectively, improved tolerance to endosulfan with growth. The POD activities of test organisms exposed to low concentrations of endosulfan were inhibited, indicating the presence of oxygen damaged tissue. Moreover, a notable decrease in AChE activity was observed due to overstimulation of neurotransmission, which might result in abnormal behavior. The effect caused by endosulfan on phosphatase production in the hepatopancreas of prawns 1.5, 4, and 7 cm long was different because the ability of nonspecifi c immune regulation increased with growth. The 96 h LC 50 values obtained in this study could be used in the formulation of water-quality criteria in China. Moreover, the changes in enzymes activities of M. rosenbergii under stress of endosulfan could be applied in the establishment of early warning indicators for bio-safety.

Histochemical study of the pre- and postnatal development of acetylcholinesterase in the rat spinal cord

The distribution of acetylcholinesterase(AChE)-positive structures in the developing rat spinal cord was studied with AChE-histochemistry.AChE-positive perikarya were first seen on embryonic day 14(E14) in the ventrolateral portion of the spinal cord.From that time onward.AChE=containing cells appeared gradually in the intermediate gray,dorsal horn and lateral spinal nucleus of the spinal cord in a ventral-to-dorsal,and lateral-to-medial order.No obvious rostral-to-caudal sequence was found.At birth,the distribution pattern of AChE-positive perikarya was basically similar to that in adults.After birth a dramatic increase in the AChE staining intensity extended from postnatal day 5(P5) to postnatal day 21(P21),In addition,two phases of transient AChE staining were observed in the external surface of the dorsal horn from embryonic day 15(E15) to embryonic day 21(E21) and in the marginal layer from embryonic day 21(E21) to postnatal day 14(P14),respectively.

Acetylcholinesterase inhibitory activities of 48 traditional Chinese medicinal herbs

Inhibition of acetylcholinesterase （ACHE） is one of the approaches for the treatment of Alzheimer＇s disease （AD）. In this paper the AChE inhibitory activities of ethanolic and aqueous extracts of 48 traditional Chinese medicinal herbs were evaluated. These traditional Chinese medicines have intelligence-promoting, anti-insomnia, sedative, neurotonic or tonic effects. Microplate assay indicated that ethanolic extracts of 28 traditional Chinese medicinal herbs and water extracts of 11 herbs showed AChE inhibitory activities. Among them, ethanolic extracts of Herba Moslae, Fructus Alpiniae Oxyphyllae, Radix Rehmanniae and Folium Nelumbinis showed most potent AChE inhibitory activities at the concentration of 0.1 mg/mL, with （68.63±1.12）%, （44.49±3.66）%, （43.78±4.76）%, and （42.63±8.31）% inhibition, respectively. The AChE inhibitory activities were also confirmed by TLC bioautographic assay. These results partially validate the traditional uses of some medicinal herbs for cognitive improvement.

In vitro studies on the multi-target anti-Alzheimer activities of berberine-like alkaloids from Coptidis Rhizoma

Coptidis Rhizoma, a well known heat-clearing Chinese Materia Medica, exhibited multi-target anti-Alzheimer effectsin vitro through inhibition of AI342 aggregation, ACHE, and BACE1. Six berberine-like alkaloids were found to be the majoractive constituents of Coptidis Rhizoma by HPLC analysis along with activity assays. A standard combination of the six majoralkaloids was demonstrated to fully reproduce the in vitro effects of Coptidis Rhizoma. Molecular docking studies revealed thatberberine-like alkaloids could directly bind at the active sites of AI342, ACHE, and BACE1 through hydrophobic interactions,aromatic stacking, and electrostatic interactions.

Multiple biological functions and pharmacological effects of lycorine

Lycorine is the major active component from the amaryllidaceae family plant Lycoris radiate,a represent traditional Chinese medicinal herb,and is one of the typical alkaloids with pyrrolophenanthridine nucleus core.Lycorine has drawn great interest in medicinal field due to its divergent chemical structures and multiple biological functions,as well as pharmacological effects on various diseases.Accumulated evidence shows that lycorine not only possesses strong pharmacological effects on many diseases,including anti-leukemia,anti-tumor,anti-angiogenesis,anti-virus,anti-bacteria,anti-inflammation,and anti-malaria,but also exerts many other biological functions,such as inhibition of acetylcholinesterase and topoisomerase,suppression of ascorbic acid biosynthesis,and control of circadian period length.Notably,lycorine exhibits its numerous pharmacological effects on various diseases with very low toxicity and mild side effects.The divergent chemical structures,multiple biological functions,and very low toxicity of lycorine imply that the agent is a potential drug candidate that warrants for further preclinical and clinic investigation.

Therapeutic effects of human amniotic epithelial cell transplantation on double-transgenic mice co-expressing APPswe and PS1ΔE9-deleted genes

Human amniotic epithelial cells (HAECs), which exhibit characteristics of embryonic and pluripotent stem cells, could be utilized for cell therapy without legal or ethical problems. Double-transgenic (TG) mice (n=20) and wild-type (WT) mice (n=20) were randomly assigned to two groups, respectively. The transplantation group was treated with HAECs and the control group with PBS. A six-radial arm water maze was used to assess spatial memory. Immunofluorescence was utilized to track HAEC survival. Immunohistochemistry was used to determine octamer-binding protein 4 (oct-4) and nanog expression in the HAECs. High-performance liquid chromatography (HPLC) was used to measure acetylcholine levels in the hippocampus. The density of cholinergic neurons in the basal forebrain and nerve fibers in the hippocampus was measured following acetylcholinesterase staining. Results showed that transplanted HAECs survived for at least eight weeks and migrated to the third ventricle without immune rejection. Graft HAECs also expressed the specific stem cell markers oct-4 and nanog. Compared with the control group, HAEC transplantation significantly ameliorated spatial memory deficits in TG mice, as well as increased acetylcholine levels and the number of hippocampal cholinergic neurites. Intracerebroventricular HAEC transplantation improved spatial memory in double-TG mice, and results suggested that increased acetylcholine levels in the hippocampus, released by surviving cholinergic neurites, were responsible for this improvement.

Ameliorating effect of Zhizi(Fructus Gardeniae) extract and its glycosides on scopolamine-induced memory impairment

OBJECTIVE： To study the chemical constituents of Zhizi （Fructus Gardeniae） and their antiamnesic ef- fect in a mouse model of Alzheimer＇s disease. METHODS： Ameliorating effects of the extracts, fractions and constituents on scopolamine-in- duced memory impairment in vivo using a passive avoidance task system and their inhibitory activi- ties on acetylcholinesterase （ACHE） in vitro were ex- amined. The isolation of components was per- formed by chromatographic techniques and their structures were identified on the basis of spectral analysis. RESULTS： Activity-guided fractionation of the total extracts resulted in the isolation of two glycosides, geniposide and cr0cin from the n-butanol fraction and genipin and crocetin from the ethylacetate fraction. Among the fractions tested, n-butanol fraction showed the strongest AChE inhibition （43.4% at a final dose of 0.03 mg/mL） and also ex- hibited outstanding efficacy （65.9% at a dose of2.50 mg/kg） in an experimental model of amnesia. Geniposide showed a 22.8% AChE inhibitory activi- ty and a potent ameliorating effect on scopol- amine-induced memory impairment in amnesic mice of 93.4% as compared to the control group. CONCLUSION： Geniposide, a main constituent of gardenia should be considered a candidate for fur- ther clinical study for the purpose of developing a cognition activator and its mechanism of action may be mediated, at least in part, by the acetylcho- line enhancing cholinergic nervous system.

Probing enzyme-nanoparticle interactions using combinatorial gold nanoparticle libraries

The interaction of nanoparticles with proteins is extremely complex, important for understanding the biological properties of nanomaterials, but is very poorly understood. We have employed a combinatorial library of surface modified gold nanoparticles to interrogate the relationships between the nanoparticle surface chemistry and the specific and nonspecific binding to a common, important, and representative enzyme, acetylcholinesterase （ACHE）. We also used Bayesian neural networks to generate robust quantitative structure-property relationship （QSPR） models relating the nanoparticle surface to the AChE binding that also provided significant understanding into the molecular basis for these interactions. The results illustrate the insights that result from a synergistic blending of experimental combinatorial synthesis and biological testing of nanoparticles with quantitative computational methods and molecular modeling.

Ameliorating effects of constituents from Cortex Acanthopanacis Radicis on memory impairment in mice induced by scopolamine

OBJECTIVE:To investigate the effects of active constituents extracted from Cortex Acanthopanacis Radicison improving the impaired memory in mice models.METHODS:The mice models of memory impairment were established using scopolamine.Ameliorating effects of the fractions and constituents on scopolamine-induced memory impairment in vivo were investigated using passive avoidance and Morris water-maze task tests,and their anti-acetylcholinesterase(AChE)and antioxidant activities in vitro examined.The isolation of constituents was performed by chromatographic methods and their structures were identified on the basis of instrumental analysis.RESULTS:Among the fractions tested,ethylacetate fraction exhibited the anti-AChE activity(25.83%±0.23%)properly and excellent 2,2-diphenyl-1-picrylhydrazyl(DPPH)radical and superoxide anion scavenging capacity(87.50%±0.83%and 60.22%±0.43%,respectively).However,the methylene chloride fraction was much more active than the ethylacetate fraction in the passive avoidance task test(167.5%increase of step-through latency time)and Morris water-maze task test(33.3%decrease of es-cape latency time).Four constituents,β-sitosterol,stigmasterol,sesamin,and hyperin were isolated from the methylene chloride fraction,among them,hyperin showed anti-acetylcholinesterase and anti-oxidant activities remarkably.Moreover,hyperin exerted a potent effect(146±38)s on memory improvement in terms of passive avoidance task test compared with the reference compound tacrine(162±43)s at a dose of 2.5 mg/kg.CONCLUSION:Hyperin,a flavonoid glucoside isolated from Cortex Acanthopanacis Radicis,inhibited AChE activity and potently ameliorated scopolamine-induced memory impairment,and its action may be partially mediated by the acetylcholine-enhancing cholinergic nervous system.

Acetylcholinesterase liquid crystal biosensor for identification of AChE inhibitors by a reactivator

An improved acetylcholinesterase liquid crystal(LC) biosensor has been developed for the identification of organophosphates(OPs) by using a reactivator. When the acetylcholinesterases(AChEs) inhibited by different kinds of OPs are reactived by a reactivator, the catalytic activity of AChEs can be recovered with different activation efficiency because of the different phosphorylation structures formed in the inhibited AChEs. Accordingly, the reactived AChEs can catalyze the hydrolysis of acetylthiocholine to generate thiocholine product in different degrees, which will result in different catalytic growth of AuNPs and further form distinct orientational response of LCs. Based on such a reactivation mechanism, the AChE LC biosensor with a simple, rapid and visual procedure achieves an obvious identification of three OPs pesticides, methamidophos, trichlorfon and paraoxon, by using a pralidoxime reactivator.

Design, synthesis and biological evaluation of novel quinoline [4,3-b] chromene derivatives as AChE inhibitors through an efficient one-pot, four-component microwave-mediated reaction

An efficient synthesis of chromeno[4,3-b]quinoline derivatives via one-pot,four-component reaction of 4-hydroxycoumarin, formaldehyde,cyclohexanedione,ammonium ceric nitrate under microwave irradiation was accomplished.The structures of these compounds were unambiguously confirmed by single crystal X-ray diffraction.Furthermore,the anti-AChE activities of these compounds in vitro were investigated at concentrations of 20μM and 50μM by using a standard Ellman's method.The relationship of inhibitory activities and structures of these chromeno [4,3-b]quinolines was also systematically studied.Of all the compounds investigated,4ag emerged as the most potent AChE inhibitor with IC50 values of 5.63μM,and it might be used as potent lead for the development anti-AChE agents.Moreover,molecular modelling was conducted to understand the optimal interaction of AChE with these types of compounds.