Acute effects of chlorpyryphos-ethyl and secondary treated effluents on acetylcholinesterase and butyrylcholinesterase activities in Carcinus maenas

The acute effects of commercial formulation of chlorpyrifos-ethyl （Dursban ） and the secondary treated industrial/urban effluent （STIUE） exposure on acetylcholinesterase （ACHE） and butyrylcholinesterase （BuChE） activities in hepatopancreas and gills of Mediterranean crab Carcinus maenas were investigated. After 2 d of exposure to chlorpyriphos-ethyl, the AChE activity was inhibited in both organs at concentrations of 3.12 and 7.82 μg/L, whereas the BuCHE was inhibited only at higher concentration 7.82 μg/L of commercial preparation Dursban~. The exposure of crabs to Dursban （3.12 μg/L） showed a significant decrement of ACHE activity at 24 and 48 h, whereas the BuChE was inhibited only after 24 h and no inhibition for both enzymes was observed after 72 h. Moreover, a significant repression of AChE activity was observed in both organs of C. maenas exposed to 5% of STIUE. Our experiments indicated that the measurement of AChE activity in gills and hepatopancreas of C. maenas would be useful biomarker of organophosphorous （OP） and of neurotoxic effects of STIUE in Tunisia.

Prevalence of Wild-Type Butyrylcholinesterase Genotype in Patients with Alzheimer’s Dementia

Approximately, two-thirds patients with Alzheimer’s disease (AD) are reported to have homozygous wild-type butyrylcholinesterase (BuChE) gene expression. It is associated with a higher rate of hydrolysis of acetylcholine, which ultimately leads to increase in the levels of BuChE in advanced stages of the disease. Rivastigmine, a dual inhibitor of acetylcholinesterase (AChE) and BuChE, might be of additional benefit in patients with AD with wild-type BuChE allele.

Expression and characterization of a codon-optimized butyrylcholinesterase for analysis of organophosphate insecticide residues

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.

A Practical and High-Affinity Fluorescent Probe for Butyrylcholinesterase:A Good Strategy for Binding Affinity Characterization

Butyrylcholinesterase(BChE)is regarded as a promising target for the treatment of Alzheimer's disease(AD),as its level significantly increases along with the progress of this disease.Therefore,the development of potent and high-affinity small-molecule BChE inhibi-tors may be a new strategy for the discovery of anti-AD drugs.However,the current Ellman's method is unable to evaluate the affinity of compounds with BChE,and has a few deficiencies in drug development.Herein,the first small-molecule fluorescence polarization(FP)probes for BChE were rationally designed based on a high affinity inhibitor.Studies indicated that probe F6 exhibited satisfactory fluorescence intensity and suitable fluorescent properties that were compatible with the filters in the FP system.Meanwhile,probe F6 exhibited potent binding affinity to BChE.It is feasible to be applied in detecting the affinity of non-fluorescent compounds to BChE,which lays a solid foundation for the development of small-molecule BChE inhibitors.At the same time,it also can be applied as a val-uable chemical tool for better understanding the molecular biological mechanism of BChE.

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alternative method to produce this protein.One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE.In this study,we cloned the hBCHEgene into a human lactoferrin(hLF)bacterial artificial chromosome(BAC)construct to make a hLFhBCHE BAC construct.Subsequently,we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice.Expression analysis showed that recombinant hBCHE(rhBCHE)was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 76
12 to 159
28 mg·L^(–1).Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE.Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.

Emerging links between type 2 diabetes and Alzheimer's disease

Type 2 diabetes mellitus and Alzheimer's disease are both associated with increasing age,and each increases the risk of development of the other.Epidemiological,clinical,biochemical and imaging studies have shown that elevated glucose levels and diabetes are associated with cognitive dysfunction,the most prevalent cause of which is Alzheimer's disease.Cross sectional studies have clearly shown such an association,whereas longitudinal studies are equivocal,reflecting the many complex ways in which the two interact.Despite the dichotomy,common risk and etiological factors(obesity,dyslipidemia,insulin resistance,and sedentary habits) are recognized;correction of these by lifestyle changes and pharmacological agents can be expected to prevent or retard the progression of both diseases.Common pathogenic factors in both conditions span a broad sweep including chronic hyperglycemia per se,hyperinsulinemia,insulin resistance,acute hypoglycemic episodes,especially in the elderly,microvascular disease,fibrillar deposits(in brain in Alzheimer's disease and in pancreas in type 2 diabetes),altered insulin processing,inflammation,obesity,dyslipidemia,altered levels of insulin like growth factor and occurrence of variant forms of the protein butyrylcholinesterase.Of interest not only do lifestyle measures have a protective effect against the development of cognitive impairment due to Alzheimer's disease,but so do some of the pharmacological agents used in the treatment of diabetes such as insulin(especially when delivered intranasally),metformin,peroxisome proliferator-activated receptors γ agonists,glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors.Diabetes must be recognized as a risk for development of Alzheimer's disease;clinicians must ensure preventive care be given to control and postpone both conditions,and to identify cognitive impairment early to manage it appropriately.

Awareness during emergence from anesthesia: Features and future research directions

The anesthesia awareness with recall(AAWR) phenomenon represents a complication of general anesthesia consisting of memorization of intraoperative events reported by the patient immediately after the end of surgery or at a variable distance from it. Approximately 20% of AAWR cases occur during emergence from anesthesia. Clinically, these unexpected experiences are often associated with distress especially due to a sense of paralysis. Indeed, although AAWR at the emergence has multiple causes, in the majority of cases the complication develops when the anesthesia plan is too early lightened at the end of anesthesia and there is a lack of use, or misuse, of neuromuscular monitoring with improper management of the neuromuscular block. Because the distress caused by the sense of paralysis represents an important predictor for the development of severe psychological complications, the knowledge of the phenomenon, and the possible strategies for its prophylaxis are aspects of considerable importance. Nevertheless, a limited percentage of episodes of AAWR cannot be prevented. This paradox holds also during the emergence phase of anesthesia which represents a very complex neurophysiological process with many aspects yet to be clarified.

Phytochemical composition of Caesalpinia crista extract as potential source for inhibiting cholinesterase and β-amyloid aggregation: Significance to Alzheimer's disease

Objective: To screen plant extract fractions and elucidate the components present in Caesalpinia crista(C. crista) leaves for cholinergic and anti-amyloidogenic activities for the treatment of Alzheimer's diseases. Methods: This work has been carried out to study the action of C. crista extracts from nonpolar to polar solvents toward inhibition of oxidative stress, cholinergic and amyloidosis. The antioxidant activity was studied using DPPH total antioxidant assay; cholinergic assay by Ellman's method and anti-amyloidogenic assay by thioflavin-T fluorescence and transmission electron microscopy. Results: The quantification of polyphenols was carried out following C. crista methanolic extract(CCMeOH) HPLC fingerprinting, along with LC-MS and elucidated by MS LAMPS database. GC-MS of CCMeOH was screened for potential moieties. In vitro experimental results showed that the CCMe OH was potential extract that exhibited active inhibition of antioxidant property, cholinergic enzymes acetylcholinesterase and butyrylcholinesterase. For anti-amyloidogenic evaluations, among all the extracts, the CCMe OH was found to have the potential toward inhibiting the oligomers, fibrillation of Aβ42 with good defibrillation of amyloid cascading properties. Conclusions: These results are also supported by the presence of polyphenols as the active ingredients. Multi-potent target drug therapy is a promising option in treating the Alzheimer's diseases. Methanolic extract of C. crista shows potential activity against cholinergic enzymes, Aβ42 aggregation with antioxidant activity.

8-Hydroxyquinolylnitrones as multifunctional ligands for the therapy of neurodegenerative diseases

We describe the development of quinolylnitrones(QNs)as multifunctional ligands inhibiting cholinesterases(ChEs:acetylcholinesterase and butyrylcholinesterase—h BChE)and monoamine oxidases(hMAO-A/B)for the therapy of neurodegenerative diseases.We identified QN 19,a simple,low molecular weight nitrone,that is readily synthesized from commercially available 8-hydroxyquinoline-2-carbaldehyde.Quinolylnitrone 19 has no typical pharmacophoric element to suggest ChE or MAO inhibition,yet unexpectedly showed potent inhibition of h BChE(IC50=1.06±0.31 nmol/L)and h MAO-B(IC_(50)=4.46±0.18μmol/L).The crystal structures of 19 with hBChE and hMAO-B provided the structural basis for potent binding,which was further studied by enzyme kinetics.Compound 19 acted as a free radical scavenger and biometal chelator,crossed the blood—brain barrier,was not cytotoxic,and showed neuroprotective properties in a 6-hydroxydopamine cell model of Parkinson's disease.In addition,in vivo studies showed the anti-amnesic effect of 19 in the scopolamine-induced mouse model of AD without adverse effects on motoric function and coordination.Importantly,chronic treatment of double transgenic APPswe-PS1δE9 mice with 19 reduced amyloid plaque load in the hippocampus and cortex of female mice,underscoring the disease-modifying effect of QN 19.

In vitro antioxidant assessment and a rapid HPTLC bioautographic method for the detection of anticholinesterase inhibitory activity of Geophila repens

OBJECTIVE： Geophila repens （L.） I. M. Johnst. （Rubiaceae）, a small, creeping, perennial herb, is claimed to have memory-enhancing property. The goal of this study was to assess its antioxidant and anticholinesterase activity and conduct a rapid bioautographic enzyme assay for screening acetylcholinesterase （ACHE） and butyrylcholinesterase （BChE） inhibition of G. repens extracts. METHODS： Antioxidant activity of G. repens extracts was assessed by performing 1,1-diphenyl-2- picrylhydrazyl （DPPH）, nitric oxide （NO）, superoxide （SOD）, hydroxyl （OH） and total antioxidant capacity （TAC） assays. Anticholinesterase activity was investigated by quantifying the AChE and BChE inhibitory activities of chloroform （CGR）, ethyl acetate （EGR） and methanol （MGR） extract fractions from G. repens leaves. A rapid high-performance thin-layer chromatography （HPTLC） bioautographic method for the detection of AChE and BChE inhibition was performed. RESULTS： Among all extract fractions, EGR exhibited the highest half maximal inhibitory concentration （IC50） in DPPH, SOD, NO, OH and TAC assays, with IC50 of （38.33 ± 3.21）, （45.14 ±1.78）, （59.81± 1.32）, （39.45 ± 0.79） and （43.76 ± 0.81 ） μg/mL respectively. EGR displayed competitive, reversible inhibition of AChE and BChE activities with IC50 of （68.63±0.45） and （59.45 ±0.45）μg/mL, respectively. Total phenolic and flavonoids contents of EGR were found to be 360.42 mg gallic acid equivalents and 257.31 mg quercetin equivalents per gram of extract. Phytoconstituents of the EGR extract that were inhibitors of cholinesterase produced white spots on the yellow background of HPTLC plates in the bioautographic test. CONCLUSION： The results of this study revealed that phenols and flavonoids could be responsible for the antioxidant, anticholinesterase activities of G. repens.

Discovery of Tryptophan-tetrahydroisoquinoline Derivatives as Multifunctional Agents for Treatment of Alzheimer's Disease

Comprehensive Summary The cholinesterases are essential targets implicated in the pathogenesis of Alzheimer's disease(AD).We have identified tryptophan-tetrahydroisoquinoline derivatives as selective micro-nanomolar butyrylcholinesterase(BChE)inhibitors.Molecular docking was applied for the rational design and binding mode analysis.They were defined according to their target inhibitory activity,low cytotoxicity,predicted permeability through the blood-brain barrier(BBB),and in vivo cognitive improvement.Additionally,the preferred compound showed ability to decrease self-induced Aβ1-42 aggregation and Aβ1-42 induced SH-SY5Y cell injury.Altogether,these factors indicated their potential as unique lead compounds for AD treatment.