鲮鱼冷休克及其死亡的某些生化因素

采用分光光度法和淀粉凝胶电泳法初步研究了鲮鱼冷休克前后脑乙酰胆碱酯酶(AchE)活力和三种酶的同工酶类的动态变化。并根据鲮鱼在冷休克期间(7—6℃)脑AchE活力显著降低和肝脏组织乳酸脱氢酶(LDH)同工酶活性明显升高以及苹果酸脱氢酶(MDH)、酯酶(EST)同工酶类出现酶活性变化的情况,讨论了导致鲮鱼耐寒能力差的某些生化因素。同时提出脑AchE可以作为评定鲮鱼冷休克期间中枢神经系统受害程度的一种生化指标。       

CH/pi hydrogen bonds as evidenced in the substrate specificity of acetylcholine esterase

The crystal structure of acetylcholine esterase (AchE) in complex with various inhibitors, investigated as drugs for improvement of the cognitive ability of early stage Alzheimer's disease, has been analyzed with the use of our program CHPI. A number of CH/pi hydrogen bonds have been disclosed in the binding of the inhibitors with Torpedo californica AchE. It has been demonstrated that, in order to be effective in the binding with AchE, C-H bonds in the inhibitor need not be polarized.     

Evidence for a striking increase in acetylcholinesterase activity in cultured human fibroblasts which are trisomic for chromosome two

Acetylcholinesterase (AchE) is reported to have a narrowly restricted distribution among human tissues. Three strains of human fibroblasts which are trisomic for chromosome 2 had an average level of AchE activity over 28 times higher than the average level in 19 control strains of human fibroblasts. In contrast, the mean pseudocholinesterase activity of the trisomy-2 strains did not differ appreciably, or significantly, from the mean for the control strains. The 19 control strains included 10 strains trisomic for autosomes other than 2, and 9 euploid strains. Our estimate of the mean AchE activity in the control strains did not differ significantly from zero and might, in any case, have originated from a minute amount of activity contributed to the cells by an esterase in our culture medium. Despite the striking elevation of AchE activity in fibroblasts trisomic for chromosome 2, extracts of these cells had only about 1.5% of the specific AchE activity (per microgram DNA) present in extracts of human cerebral cortex. None of the 22 strains studied had detectable activity for two other enzymes which, like AchE, have a restricted distribution among human tissues: xanthine oxidase and choline acetyltransferase.     

Molecular form of human lymphocyte membrane-bound acetylcholinesterase

The membrane-bound acetylcholinesterase (AchE) from human peripheral blood lymphocyte gives only one symmetrical peak on sucrose density gradient centrifugation in the presence of Triton X-100 detergent, with the calculated sedimentation coefficient of 6.5 S. However, this dimeric form of AchE was converted to a monomeric 3.8 S form when treated with 2-mercaptoethanol and iodoacetic acid. The results are consistent with studies which have shown by sodium dodecyl sulfate gel electrophoresis that the enzyme is built up of two identical monomers inter-linked by disulfide bond(s). Under reducing conditions, revealed a single species of 70,000 molecular weight, whereas under non-reducing conditions, another species of 140,000 molecular weight of the AchE was found. Polyacrylamide gel electrophoresis indicated a single band with AchE activity in the presence of Triton X-100. In contrast, in the absence of the same detergent multiple band pattern could be observed. These results suggest that membrane-bound AchE enzyme is present in homogenous dimeric form on human lymphocyte membrane.     

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage.
Purification of AchE from plutei has been carried out by the procedure including affinity chromatography. Purified AchE had the activity 14,600 times higher than that of homogenate, and the final yield of AchE was 8%. The enzyme seems to be electrophoretically homogeneous, and has a molecular weight of 3 × 10⁵ as determined by Sepharose CL–6B column chromatography.     

Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase

A method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP-treated horse serum +/- pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) in 96-well microplates. Following the culture period, Triton X-100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well. AchE synthesized in culture cleaved acetylthiocholine to thiocholine, which stochiometrically reduced the colorless indicator DTNB to a highly colored product. Thirty minutes following the addition of substrate, the plates were assayed for activity with a vertical recording photometer. When platelets, freshly prepared bone marrow cells, or cultured marrow were assayed by this method, a linear relationship was observed between optical density (OD) and the number of cells assayed. Moreover, a linear relationship between the number of AchE-positive megakaryocytes determined histochemically and AchE activity determined spectrophotometrically was observed. Red cells exhibited no activity. Inhibitor studies demonstrated that the activity measured was true AchE. Separation of marrow by density gradient centrifugation showed that the megakaryocyte enriched fraction contained all the AchE while the megakaryocyte depleted fraction contained none. From the data we conclude that this rapid, semiautomated method quantitates megakaryocytic AchE synthesis in culture, and that this method will be a useful assay system for the detection of factors that influence megakaryocytopoiesis.     

Changes in acetylcholinesterase during pupal development of Apis mellifera queen

Total, membrane, and soluble acetylcholinesterase (AchE, EC 3.1.1.7) activities increase during the pupal development of Apis mellifera queen to reach maximum values at emergence. Membrane and soluble AchE are inhibited by 10^-5 M eserine or BW284C51 except at Pr, Pdm, and Pdd stages in which soluble AchE presents eserine-sensitive and eserine-resistant fractions. At all pupal stages, AchE occurs in a major amphiphilic membrane form that represents about 98% of total AchE activity and whose sedimentation coefficient is about 5.7S, and in a minor hydrophilic form that represents about 2% of total AchE activity and whose sedimentation coefficient is about 7S. At all pupal stages, phosphatidylinositol-specific phospholipase C (PI-PLC) and glycosyl phosphatidylinositol-specific phospholipase D (GPI-PLD) convert the membrane form into soluble counterparts which electrophoretic mobilities differ from that of the soluble form. AchE exhibits a butyrylcholinesterase (BuChE) activity that represents about 14% of AchE activity. During pupal development, the BuChE/AChE ratio of the membrane fraction is relatively stable, whereas the BuChE/AChE ratio of the soluble fraction is subjected to significant variations. At early pupal stages (Pw–Pd), membrane AchE displayed a high K_m value, higher than 40 μM, that decreases to an intermediary value of about 30 μM at Pdl and Pdm stages, to reach finally about 20 μM at Pdd and emergence stages. Arch. Insect Biochem. Physiol. 36:69–84, 1997. © 1997 Wiley-Liss, Inc.     

论淡色库蚊敌百虫抗性品系的抗药性机理

分别测定了淡色库蚊(Culex pipiens pallens)敏感品系(SEN)及敌百虫抗性品系(RD)雌成蚊的乙酰胆碱酯酶(AchE)对硫代乙酰胆碱(Atch)的米氏常数(K_m)和最大反应速度(V_max).SEN雌成蚊头部AchE的K_m=1.2×10^-4mol/L,V_max=7.7×10^-3mol/L(产物);胸部的K_m=0.8×10^-4mol/L,V_max=6.5×10^-3mol/L(产物).RD雌成蚊头部AchE的K_m=5.7×10^-4mol/L,V_max=25×10^-3mol/L(产物);胸部的K_m=0.8×10^-4mol/L,V_max=9.5×10^-3mol/L(产物).结果表明:1.SEN和RD二者的雌成蚊,它们自身头、胸间AchE是不同质的,是以同工酶的形式存在,因此RD雌成蚊头部AchE的性质与SEN雌成蚊的头部有显著差异也是由AchE同工酶的变异所造成.2.RD雌成蚊头部AchE在量上与SEN间也存在明显的差异.因此可以认为RD雌成蚊对有机磷产生抗性的主要机理是头部AchE在质和量上产生了变异.由于羧酸酯酶不是对具有磷酸酯键的有机磷制剂作用的靶子酶,因而不是敌百虫抗性产生的主要原因.     

双酚A暴露对东亚三角涡虫急性毒性及神经酶的影响

旨在探讨双酚A (BPA)对东亚三角涡虫的急性毒性及神经系统相关酶活性的影响。采用不同浓度的BPA处理涡虫24 h、48 h、72 h,求出半致死浓度,以此为基础,采用不同浓度的BPA处理涡虫24 h、72 h、144 h,测定AchE、ChAT及Na+~K+^-ATP酶活性。BPA对三角涡虫的24 h、48 h、72 h,LC50分别为12.18 mg/L、8.49 mg/L、6.43 mg/L。ChE、ChAT、Na+~K+^-ATP酶活性对BPA反应敏感,具有较好的规律性。24 h处理组,AChE酶活力随BPA浓度的升高而升高,72 h和144 h处理组则呈现先上升后下降的趋势,除0.643 mgBPA/L以外,其它处理组均表现出了时间-效应关系;在BPA胁迫下,ChAT酶活力均呈现降低趋势,在BPA浓度为0.643 mg/L、1.286 mg/L时,表现出了时间-效应关系;24 h、72 h处理组Na+~K+^-ATP酶活力随BPA浓度升高呈现先上升后下降的趋势,144 h处理组则呈现下降趋势,Na+~K+^-ATP酶活力随BPA胁迫时间的延长呈现先升高后下降的趋势。BPA对涡虫具有较强的生态毒性,AchE、ChAT、Na+~K+^-ATP酶活性可与其他敏感指标一起作为水体BPA污染的早期监测指标。     

Activation/deactivation of acetylcholinesterase by H2O2: more evidence for oxidative stress in vitiligo

Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3)M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6)M) activate this enzyme by increasing the Vmax>2-fold, meanwhile high concentrations of H2O2 (10(-3)M) inhibit the enzyme with a significant decrease in Vmax. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambdamax 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo.     

Clinical trials of bestatin for leukemia and solid tumors

Dissociated cells from 13- and 17-day-old embryonic rat mesencephali have grown in primary cultures in order to compare the early and late influences of different agents - insulin, dexamethasone and nerve growth factor (NGF) - on the expression of cholinergic maturation process. We have studied cholin acetyltransferase (ChAT) activity, which is regarded as a specific marker for cholinergic function of the brain, and a widely used differentiation marker, the acetyl-cholinesterase (AchE) enzyme. Biochemical maturation of increasing specific activity of ChAT in both younger and older cells was taken into consideration. During cultivation the AchE activity was slightly increased in younger cells, but a dramatic decrease could be noted in older ones. Insulin in concentration from 10 to 27 µg mL^–1 causes a significant inhibition in ChAT activity in comparison with the enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng), independently of embryos age. This polypeptide hormone is able to enhance AchE activity in the cultured cells, especially in older ones. With continuous treatment of the culture with dexamethasone, a synthetic glucocorticoid, the ChAT activity in younger cells reaches a maximum curve by day 9 (nine). At this time the AchE activity shows a slighter, no significant increase than at any other time during cultivation. In cell cultures taken from 17-day-old embryos however dexamethasone treatment evoked a significant decrease in ChAT activity with a concomitant increase of AchE activity which was compared to insulin treatment. In spite of the fact that the NGF is able to enhance the ChAT activity, no significant alteration in AchE activity can be measured in younger cell cultures. These results suggest an uneven expression of the enzymes in embryonic rat mesencephali in the presence of above agents depending on the age of cells.     

Insulin secretion and acetylcholinesterase activity in monosodium l-glutamate-induced obese mice.

OBJECTIVE: Pancreatic islets isolated from mice treated neonatally with monosodium L-glutamate (MSG) were used to study insulin secretion. MATERIALS AND METHODS: Total acetylcholinesterase (AchE) activity of tissue extract was measured as a cholinergic activity marker. Obesity recorded in 90-day-old MSG mice (OM) by Lee index reached 366.40 +/- 1.70, compared to control mice (CM) 324.40 +/- 1.10 (p < 0.0001). Glucose 5.6 mM induced insulin secretion of 36 +/- 5 pg/15 min from islets of CM and 86 +/- 13 from OM (p < 0.001). When glucose was raised to 16.7 mM, islets from OM secreted 1,271 +/- 215 and 1,017 +/- 112 pg/30 min to CM. AchE activity of pancreas from OM was 0.64 +/- 0.02 nmol of substrate hydrolyzed/min/mg of tissue and 0.52 +/- 0.01 to CM (p < 0.0001). Liver of obese animals also presented increase of AchE activity. RESULTS: These indicate that OM insulin oversecretion in low glucose may be attributed, at least in part, to an enhancement of parasympathetic tonus.     

The Role of Erythrocytic Acetylcholinesterase in Hormonal Regulation of Adaptation to Physical Exercise

Acetylcholinesterase (AchE) activity in erythrocytes and blood levels of cortisol and insulin were investigated in athletes training under different bioenergetic conditions (sprinters, middle-distance runners, and marathoners). The groups of sprinters and marathoners had a decreased enzyme activity compared to nonathletes (p < 0.05). In response to a standard exercise, load AchE activity increased in the groups of middle-distance runners and marathoners. A relationship was observed between the level of AchE activity and the cortisol-to-insulin ratio in the blood. This ratio is specific to the type of bioenergetic conditions and increases in the following order: controls, middle-distance runners, sprinters, and marathoners. In vitro experiments revealed an effect of insulin on AchE activity. This effect was significantly lower in sprinters than in the control group. A reduction in AchE activity and an increase in the cortisol-to-insulin ratio are considered as factors increasing metabolic turnover in athletes, mainly, lipid turnover. This mechanism ensures effective mobilization of substrates at the start of physical exercise and their recovery after. The observed relationship between the insulin level and the AchE activity may prove to be a mechanism of regulation of the insulin level. This relationship may change during adaptation to physical exercise, as in the case of sprinters, when the sensitivity of AchE to the inhibitory effect of insulin is decreased. A high blood level of cortisol and insulin is a distinctive feature of sprinters, which provides for a higher turnover of carbohydrates. In marathoners, low AchE activity leads to an increased effect of acetylcholine, which is manifested by an increased cortisol level and a decreased insulin level, thus providing for a higher lipid turnover.     

Effect of stress on the acetylcholinesterase activity of the hypothalamus-pituitary-adrenal axis in the rat

Acetylcholinesterase (AchE, EC 3.1.1.7) activity was determined in cerebral cortex, hypothalamus, adenohypophysis and adrenal gland in response to acute and chronic stress. Chronic exposure of animals to cold stress (at 4 degrees C for 7 days) resulted in significant decline of AchE activity in all tissues studied. Similar results were obtained when animals were exposed to acute immobilization and cold stress (at 4 degrees C) simultaneously. In another experiment, animals were treated with 2 mg/kg of corticosterone prior to AchE determination. Corticosterone administration resulted in a significant decline in AchE activity of the cortex, the hypothalamus and the adrenal but failed to affect the adenohypophysis AchE level. Exposing adrenalectomized animals to acute stress resulted in no significant changes in the cortex and the hypothalamus but caused a significant decline in AchE of the adenohypophysis. It was concluded from this study that corticosterone might mediate the stress effect on AchE activity.     

Chiral nature of covalent methylphosphonyl conjugates of acetylcholinesterase

This paper examines the chiral nature of the covalent conjugates formed upon reaction of acetylcholinesterase (AchE) with enantiomeric cycloheptyl, isopropyl, and 3,3-dimethylbutyl methylphosphonyl thiocholines. With the exception of the conjugate formed from reaction of AchE with RP-cycloheptyl methylphosphonyl thiocholine, all enantiomeric conjugates underwent oxime reactivation at rates that were within 2-3-fold of each other. Oxime reactivation was, therefore, independent of both initial configuration about phosphorus and the alkyl phosphonyl ester (-OR) moiety. Aging of the enantiomeric cyclopheptyl and isopropyl methylphosphonyl conjugates occurred exclusively for the conjugate formed from the SP-enantiomer and therefore displayed an absolute dependence on the initial configuration of the methylphosphonyl group. Equilibrium titrations with decidium, a fluorescent bisquaternary competitive inhibitor of AchE, provided an index of aging and enantiomeric configuration of the conjugates independent of enzyme activity. Decidium association with the enantiomeric conjugates (prior to aging) showed no marked dependence on the initial configuration about phosphorus but was measurably dependent on nature of the -OR moiety. These results are interpreted with respect to symmetry and nonrigidity of the organophosphonyl conjugates and are consistent with formation of final methylphosphonyl conjugates that are enantiomerically pure and of opposite configuration. These studies indicate that the active center of AchE comprises at least two kinetically distinct environments separate from the esteratic region but located within 5 A of the nucleophilic serine and differing in dipolar characteristics that promote charge separation and general acid catalysis.     

Does preconception paternal exposure to a physiologically relevant level of bisphenol A alter spatial memory in an adult rat?

Bisphenol A (BPA) is a ubiquitous environmental endocrine disrupting compound (EDC); public health concerns have been fueled by findings that maternal BPA exposure can change sex differences in the brain and in some behaviors. We investigated whether a physiologically relevant dose of BPA ingested by male rats before conception would affect spatial memory and hippocampal acetylcholinesterase (AchE) in their adult offspring. Twenty-two 60-day-old male rats (F0) received either a BPA diet (50 μg/kg/day) or vehicle alone for 10 weeks before being mated with non-exposed females. The paternal rats and their forty adult offspring's (F1) behaviors were then examined in the Morris Water Maze (MWM) and their AchE activities in the hippocampus were evaluated. BPA exposure led to spatial memory deficits along with decreased AchE activities in the hippocampus (p = 0.01) in adult F0 rats. This paternal exposure also induced impairment in spatial memory acquisition in both sexes while retention only in females in F1 rats, as well as abolished sex differences in the hippocampus AchE. Overall, these data provide new evidence that paternal BPA exposure, at a “safe” dose, may induce transgenerational alterations in spatial memory in a sex-specific manner.     

Interleukin 3 promotes maturation of murine megakaryocytes in vitro

A fluorescence assay for the quantitation of acetylcholinesterase (AchE) has been adapted for measurement of megakaryocytic maturation in short-term serum-free cultures of murine marrow. When marrow cells were cultured for 3 days in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) under serum-free conditions, AchE production was found to be related to the concentration of PWM-SCM. Interleukin 3 (IL3), a purified glycoprotein promoting the proliferation of several early hematopoietic progenitors including megakaryocytic colony-forming cells, also induced AchE production in a dose-responsive manner. The response to IL3 was linearly related to the number of cells cultured. When marrow was first subjected to plastic adherence and the nonadherent cells then separated on Percoll gradients, a small megakaryocyte-enriched population markedly depleted of colony-forming cells and large megakaryocytes, responded to IL3 in a similar dose-responsive manner. A significant amount of AchE was produced in the absence of any added factors. The data show that AchE production can be measured in 3-day serum-free cultures, and suggest that IL3, a factor promoting megakaryocytic proliferation in vitro, also promotes maturation.     

Partial purification and characterization of acetylcholinesterase isozymes from adult bovine filarial parasite Setaria cervi

Filariasis is a major health problem, affecting millions of people in tropical and sub-tropical regions of the world. The isolation and characterization of parasite-specific enzyme targets is essential for developing effective control measures against filariasis. Acetylcholinesterase (AchE, E.C. 3.1.1.7), an important enzyme of neuromuscular transmission is found in a number of helminths including filarial parasites and may be playing a role in host-parasite interactions. Earlier, we demonstrated the presence of two isozymes of AchE, different from the host enzyme in the human (Brugia malayi) and bovine (Setaria cervi) filarial parasites. In the present study, two isozymes of AchE (pAchE1 and pAchE2) were isolated from S. cervi adults and characterized biochemically and immunochemically. The AchE was partially purified on Con-A Sepharose column and then subjected to preparative polyacrylamide gel electrophoresis (PAGE) for separation of the isozymes. The AchE activity was localized by the staining of gel and the isozymes were isolated from the PAGE strips by electroelution. Both isozymes preferentially utilized acetylcholine iodide as substrate and were strongly inhibited by the true AchE inhibitor (BW284c51), suggesting that they were true AchE. The polyclonal antibodies produced against the isozymes showed significant cross-reactivity with B. malayi AchE, but not against the host enzyme. These findings suggested that both the isozymes were biochemically (in terms of their substrate specificity and inhibitor sensitivity) and immunochemically similar, but different from the host enzyme.     

化湿液对湿阻证大鼠下丘脑AchE、NOS活性及NO含量的影响

目的研究化湿液对湿阻证模型大鼠下丘脑AchE、NOS活性及NO含量的影响。方法将50只SD大鼠随机分为正常组、模型组及模型组 化湿液低、中、高剂量组,每组10只。除正常组外,其余4组用改进后的环境加疲劳法制造湿阻证模型,连续造模6d。造模成功后,正常组和模型组大鼠按2ml/100g的剂量灌胃给予生理盐水;化湿液低、中、高剂量组分别按含生药0.4g/100g、0.8g/100g、1.6g/100g的剂量灌胃给予化湿液。每天1次,连续8d。取大鼠下丘脑,检测下丘脑AchE、NOS活性及NO含量。结果与正常组比较,模型组大鼠下丘脑AchE活性明显升高,NOS活性及NO含量明显降低(P<0.05);与模型组比较,化湿液可降低湿阻证模型大鼠下丘脑AchE的活性,提高下丘脑NOS活性及NO含量(P<0.05)。结论化湿液具有改善湿阻证大鼠下丘脑AchE、NOS活性及NO含量的作用,对研究化湿液治疗湿阻证的机制有一定价值。     

Butyrylcholinesterase is present in the human epidermis and is regulated by H2O2: more evidence for oxidative stress in vitiligo

The human epidermis holds the capacity for autocrine cholinergic signal transduction, but the presence of butyrylcholinesterase (BchE) has not been shown so far. Our results demonstrate that this compartment transcribes a functional BchE. Its activity is even higher compared to acetylcholinesterase (AchE). Moreover, we show that BchE is subject to regulation by H(2)O(2) in a concentration-dependent manner as it was recently described for AchE. Epidermal BchE protein expression and enzyme activities are severely affected by H(2)O(2) in vitiligo as previously demonstrated for AchE. Removal/reduction of H(2)O(2) by a pseudocatalase PC-KUS yields normal/increased protein expression and activities. H(2)O(2)-mediated oxidation of methionine residues in BchE was confirmed by FT-Raman spectroscopy. Computer simulation supported major alteration of the enzyme active site and its tetramerisation domain suggesting deactivation of the enzyme due to H(2)O(2)-mediated oxidation. Based on our results we conclude that H(2)O(2) is a major player in the regulation of the cholinergic signal via both AchE and BchE and this signal is severely affected in the epidermis of patients with active vitiligo.