棉蚜2个乙酰胆碱酯酶cDNA片段的基因克隆与序列分析

采用RT－PCR技术,利用简并引物从棉蚜（Aphis gossypii Glover)中克隆出2个乙酰胆碱酯酶基因的cDNA片段,Ag,ace 1l和Ag.ace2.Ag.ace1基因的cDNA片段为282bp,编码94个氨基酸;Ag.ace2基因的cDNA片段为264bp,编码88个氨基酸。扩增获得的2个乙酰胆碱酯酶基因cDNA片段所编码的氨基酸序列均与其他昆虫的乙酰胆碱酯酶基因有很高的同源性。首次从一种昆虫中克隆出2个乙酰胆碱酯酶基因片段,为同一种昆虫中存在多个乙酰胆碱酯酶基因的假设提供了直接的分子生物学证据。     

Development of a quantitative relationship between inhibition percentage and both incubation time and inhibitor concentration for inhibition biosensors—theoretical and practical considerations

Theoretical and practical insights into the design and development of immobilised enzyme inhibition biosensors are reported. A general mathematical expression relating the percent of enzyme inhibition (i.e. the analytical signal) to both the inhibitor concentration and the incubation time is presented. The relevant physical, chemical and biochemical parameters required by the model are developed and discussed in terms of the inhibition of acetylcholinesterase by the organophosphorous pesticide, paraoxon. A second enzyme, choline oxidase and an amperometric transducer are used to facilitate the determination acetylcholinesterase inhibitor.     

Neuroprotective role of quercetin in locomotor activities and cholinergic neurotransmission in rats experimentally demyelinated with ethidium bromide

Aim

The purpose of this study was to investigate whether the flavonoid quercetin can prevent alterations in the behavioral tests and of cholinergic neurotransmission in rats submitted to the ethidium bromide (EB) experimental demyelination model during events of demyelination and remyelination.

Main methods

Wistar rats were randomly distributed into four groups (20 animals per group): Control (pontine saline injection and treatment with ethanol), Querc (pontine saline injection and treatment with quercetin), EB (pontine 0.1% EB injection and treatment with ethanol), and EB + Querc (pontine 0.1% EB injection and treatment with quercetin). The groups Querc and Querc + EB were treated once daily with quercetin (50 mg/kg) diluted in 25% ethanol solution (1 ml/kg) and the animals of the control and EB groups were treated once daily with 25% ethanol solution (1 ml/kg). Two stages were observed: phase of demyelination (peak on day 7) and phase of remyelination (peak on day 21 post-injection). Behavioral tests (beam walking, foot fault and inclined plane test), acetylcholinesterase (AChE) activity and lipid peroxidation in pons, cerebellum, hippocampus, hypothalamus, striatum and cerebral cortex were measured.

Key findings

The quercetin promoted earlier locomotor recovery, suggesting that there was demyelination prevention or further remyelination velocity as well as it was able to prevent the inhibition of AChE activity and the increase of lipidic peroxidation, suggesting that this compound can protect cholinergic neurotransmission.

Significance

These results may contribute to a better understanding of the neuroprotective role of quercetin and the importance of an antioxidant diet in humans to provide benefits in neurodegenerative diseases such as MS.     

An acetylcholinesterase biosensor based on graphene–gold nanocomposite and calcined layered double hydroxide

In this study, a novel acetylcholinesterase-based biosensor was fabricated. Acetylcholinesterase (AChE) was immobilized onto a glassy carbon electrode (GCE) with the aid of Cu–Mg–Al calcined layered double hydroxide (CLDH). CLDH can provide a bigger effective surface area for AChE loading, which could improve the precision and stability of AChE biosensor. However, the poor electroconductibility of CLDHs could lead to the low sensitivity of AChE biosensor. In order to effectively compensate the disadvantages of CLDHs, graphene–gold nanocomposites were used for improving the electron transfer rate. Thus, the graphene–gold nanocomposite (GN-AuNPs) was firstly modified onto the GCE, and then the prepared CLDH-AChE composite was immobilized onto the modified GCE to construct a sensitive AChE biosensor for pesticides detection. Relevant parameters were studied in detail and optimized, including the pH of the acetylthiocholine chloride (ATCl) solution, the amount of AChE immobilized on the biosensor and the inhibition time governing the analytical performance of the biosensor. The biosensor detected chlorpyrifos at concentrations ranging from 0.05 to 150 μg/L. The detection limit for chlorpyrifos was 0.05 μg/L.     

Discovery of biphenyl pyrazole scaffold for neurodegenerative diseases: A novel class of acetylcholinesterase-centered multitargeted ligands

Multitargeted ligands have demonstrated remarkable efficiency as potential therapeutics for neurodegenerative diseases as they target multiple pathways involved in the progression of these diseases. Herein, we report first-in-class dual inhibitor of acetylcholinesterase (AChE) and tau aggregation as a novel class of multitargeted ligands for neurodegenerative diseases. The reported biphenyl pyrazole scaffold binds monomeric tau with submicromolar affinity and impedes the formation of tau oligomers at early stages. Additionally, the lead compound inhibited AChE activity with an IC₅₀ value of 0.35 ± 0.02 μM. Remarkably, the neuroprotective effect of this lead in induced cytotoxicity model of SH-SY5Y neuroblastoma cells is superior to single-targeted AChE and tau-aggregation inhibitors. This scaffold would enable development of new generation of multitargeted ligands for neurodegenerative diseases that function through dual targeting of AChE and monomeric tau.     

Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene

Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC₅₀ value of 34.2 nM. Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.     

乙酰胆碱酯酶在蚕豆保卫细胞中集中分布

动物细胞中,乙酰胆碱酯酶负责把乙酰胆碱水解为胆碱和乙酸以起到终止信号的作用。蚕豆（ＶｉｃｉａｆａｂａＬ．）保卫细胞原生质体表面具有特异的水解乙酰胆碱的酯酶,光可以激活该酶的活性。组织学定位的结果显示,酶反应产物主要分布于气孔保卫细胞内壁和腹壁的外侧及内壁和腹壁中,表明一种类似于动物突触传递的机制可能存在于气孔保卫细胞和周围细胞之间,即乙酰胆碱发挥作用后由乙酰胆碱酯酶分解以终止其作用;此外开放的气孔周围具有更高的酶活性,说明乙酰胆碱酯酶可通过水解气孔周围的乙酰胆碱而调控气孔运动     

Contamination of a dredged material disposal site La Rochelle Bay,France .The use of the acetylcholinesterase activity of Mytilus edulis L. as a biomarker of pesticides: the need for a critical approach

A single aspect of the toxic impact of a dredged material disposal site located near a mussel-farming zone was followed for eight months. Acetylcholinesterase activity (AChE) of Mytilus edulis was investigated as a biomarker for possible contamination by neurotoxic compounds (carbamates and/or organophosphorous pesticides). Our observations showed that the enzymatic activities (including AChE) of these harbour mussels were decreased in sites directly and indirectly influenced (according to hydrodynamic conditions) by the dumping of dredged sediments, suggesting possible contamination by pesticides. The strong correlations observed between AChE activity and growth parameters (length and weight) seems to show, however, that the enzyme activity is also indirectly controlled through growth restriction, which may imply limitation of the development of the nervous system in juveniles. The concentration of total proteins, as well as the spawning process also seem to disturb the assessment of AChE activity. These field observations clearly indicate that the use of this enzyme activity as a biomarker should proceed with caution. For example, the seasonal variability of such activity should be taken into account in a biomonitoring programme.     

Factors required for calcium dependent acetylcholine release from isolated torpedo synaptic vesicles.

The release of acetylcholine (Ach) from Torpedo synaptic vesicles has been investigated. Factors have been found which induce Ca⁺² dependent Ach release from the synaptic vesicles. In the absence of these factors, the vesicles are not affected by Ca⁺². Addition of a soluble factor to the vesicles induces a Ca⁺²-dependent release of their Ach. This secretion is enhanced by a non-vesicular membranous component which, by itself, does not induce the Ca⁺²-dependent release. These results demonstrate that vesicular Ach release may be studied $\text{in vitro}$ and thus will enable the study, at the molecular level, of the biochemical events underlying neurotransmission.     

Parasympathetic and sympathetic postganglionic synapses in ureterovesical autonomic pathways

Summary Histochemical techniques for acetylcholinesterase and catecholamine show that ureterovesical ganglia of both cat and dog contain dense intraganglionic cholinergic and adrenergic plexuses. Ramifications of both plexuses surround most cholinergic and adrenergic ganglion cell bodies as pericellular synaptic plexuses. Similar pericellular plexuses exist around extraganglionic cholinergic and adrenergic ganglion cells. Both adrenergic and cholinergic synaptic fibers persist in denervated pregnaglionic nerve-free specimens, indicating that cholinergic synaptic fibers are postganglionic parasympathetic in nature. The presence of adrenergic (postganglionic sympathetic) and postganglionic parasympathetic synapses around cell bodies in ureterovesical ganglia provides a morphologic basis for the sympathoinhibitory and muscarinic parasympatho-excitatory phenomena described in these ganglia.