不同剂量中子照射后小鼠小肠组织中乙酰胆碱和胆碱酯酶的变化

我们曾报导了不同剂量~(60)Co——丙线照射对小鼠小肠组织中乙酰胆碱含量和胆碱酯酶活性变化的影响。本文进一步观察不同剂量中子-丙线混合照射后小鼠小肠组织中乙酰胆碱和胆碱酯酶的动态变化,并和丙线照射引起的变化进行比较。以了解中子照射对小鼠小肠损伤规律及相对生物效应,研究分析中子损伤所引起的小肠运动功能紊乱及肠套叠易于发生的原因。     

pCI_(50)值测定新法及其应用

酶抑制50%时药(毒)物浓度的负对数(简称pCI_(50))是药(毒)物的重要生物活性参数.用酶活性抑制率对数与药(毒)物浓度负对数的关系计算pCI_(50)值,比用简单直线段回归及作图法结果准确,应用范围广,对低毒性药(毒)物的pCI_(50)也可计算,应用本法测定了13个新叔胺型胆碱酯酶抑制剂的pCI_(50)值,比较了小鼠脑酶与电鳐胆碱酯酶的差别.     

微量DTNB法测定胆碱酯酶活力

本文建立了微量DTNB方法测定胆碱酯酶(ChE)活力。观察了电鳐乙酰胆碱酯酶(AChE),小鼠全血ChE及小鼠脑匀浆ChE。结果表明,此方法比较灵敏,且简便、快速。明显提高了工作效率。     

人脑及红细胞膜乙酰胆碱酯酶的芳基酰胺酶活性

Fujimoto〔1〕1974年首次报道了大鼠脑中存在5-羟色胺敏感的芳基酰胺酶，认为此酶可能催化水解芳基酰胺类解热镇痛药及麻醉剂。以后又证实5-羟色胺敏感的芳基酰胺酶活性是乙酰胆碱酯酶（AchE，EC3．1．1．7）的一个属性〔2〕。通过对胆碱酯酶活性与芳基酰胺酶活性在亲和层析、凝胶过滤、电泳、抗体沉淀等方面性质的比较及底物抑制实验已确证电鳗、羊基底神经节、猴脑、人红细胞膜等来源的AChE〔3、4〕及人血清丁酰胆碱酯酶（BuchE，EC3．1．1．8）〔5〕具有芳基酰胺酶活性，此酶的特点是受5-羟色胺特异性抑制并被酪胺激活。对人血清Bu…     

苯骈咪唑酯季铵盐类的定量构效关系

采用优选结构指示变量的方法,对苯骈咪唑酯季铵盐及其抑制胆碱酯酶作用进行定量构效关系分析,获得了较为满意的结果,并对抑酶作用机理进行探讨,为合成新的胆碱酯酶抑制剂提供了参考信息。     

胆碱酯酶持续抑制下乙酰胆碱含量及拟胆碱能药物敏感性的变化

梭曼中毒难以防治的关键问题是胆碱酯酶(ACHE)磷酰化后很快老化,不易重活化。迄今为止尚未找到有效的重活化剂。值得注意的是,梭曼抑制 AChE 的程度与中毒症状的轻重有时并不呈现平行关系。如:中毒大鼠的症状有轻有重而 AChE 的抑制程度相似,或者症状业已消失但 AChE 仍被持续抑制。在人,梭曼急性中毒经阿托品及氯磷啶治疗后第三天即可起床活动而10天后红血球 AChE 仍抑制88%。众所周知,中毒后     

原花青素抗小鼠实验性血管性痴呆的研究

目的研究原花青素的抗小鼠实验性血管性痴呆作用,探讨其保护作用机制。方法采用脑缺血再灌注合并尾静脉放血降压的方法制备小鼠血管性痴呆模型,小鼠分为假手术组、模型组、阳性对照组和原花青素低、中、高（50、100、200mg.kg-1）剂量组。小鼠水迷宫实验和避暗实验用于检测空间学习记忆行为。生化测量脑内超氧化物岐化酶,胆碱酯酶的活性和总抗氧化能力、及丙二醛含量。结果原花青素低、中、高3个剂量组均可减少水迷宫逃避潜伏期时间,增加穿台次数;可显著增加小鼠被动避暗潜伏期,减少避暗穿箱次数。提高小鼠脑组织超氧化物歧化酶活性,提高总抗氧化能力,降低脂质过氧化物含量,抑制胆碱酯酶的活性。结论原花青素有抗小鼠血管性痴呆的作用,它改善血管性痴呆的机制可能与抗氧化、抑制胆碱酯酶活性有关。     

沙林及梭曼在试管内的酶促合成

沙林及梭曼的水解产物在G类毒剂水解酶催化下有外加胆碱酯酶存在时,可以生成相应的毒剂,表现为对外加胆碱酯酶活性的深度抑制。G类毒剂水解酶不能催化V类毒剂的合成。     

巨噬细胞胆碱能通路对急性缺糖缺氧性肾小管细胞损伤的影响

目的 探讨胆碱能通路对急性缺糖缺氧性肾小管细胞损伤的影响.方法 分离培养大鼠肾内巨噬细胞,构建巨噬细胞与肾小管上皮细胞共培养体系及缺糖缺氧(OGD)细胞模型,据处理不同将细胞分为OGD组、乙酰胆碱(ACh 100μmol/L)+OGD组和ACh+加兰他敏(Gal 10μmol/L)+OGD组.采用ELISA法检测各组上清液肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)和IL-10的表达;MTT法检测肾小管细胞活力;RT-qPCR及Western blotting检测胆碱酯酶(AChE)mRNA和蛋白表达;比色法检测AChE活性.结果 ACh+OGD组TNF-α和IL-1β水平均低于OGD组,加入Gal之后,TNF-α和IL-1β水平进一步下降;ACh+OGD组肾小管活力高于OGD组,加入Gal之后,肾小管活力进一步增强,差异有统计学意义(P<0.05或P<0.01).3组之间巨噬细胞AChE mRNA和蛋白表达差异均无统计学意义(P>0.05).与OGD组比较,ACh+OGD组与ACh+Gal+OGD组肾小管细胞活力减弱,但ACh+OGD组与ACh+Gal+OGD组间差异无统计学意义(P=0.368).结论 ACh和Gal可抑制肾脏巨噬细胞分泌炎性介质并抑制胆碱酯酶活性,减轻急性缺氧性肾小管细胞损伤.调控巨噬细胞的胆碱能通路可能是未来急性缺氧性肾损伤的治疗方向.     

微量乙酰靛酚比色法测量胆碱酯酶活性

微量乙酰靛酚比色法是采用脂溶性底物乙酰靛酚测量胆碱酯酶活性的方法,适用于膜结合胆碱酶、抗原抗体复合物中的胆碱酯酶等必需用脂溶性底物测定胆碱酯酶活性的实验.     

肟类药物对塔崩等神经性毒剂抑制的大鼠脑AChE的体外重活化作用

系统地研究了肟类药物对塔崩抑制的大鼠脑AChE的体外重活化作用,并与梭曼、沙林和VX进行了比较。结果表明,沙林和VX抑制的AChE较易被药物重活化,而塔崩和梭曼抑制的AChE则较难。37℃、pH7.2条件下,塔崩抑制的大鼠脑AChE可浓度依赖性地被TMB_r和LuH_6重活化,2-PAM在高浓度下也有一定作用,但HI-6在所用3个浓度下均无重活化。通过降低抑制温度成功地建立了未老化的梭曼膦酰化AChE模型。药物试验表明,未老化的梭曼膦酰化大鼠脑AChE可被高浓度(1mmol/L)HI-6重活化,而不被2-PAM、TMB_4及LuH_6重活化。提示药物自身内在活性在重活化作用中的重要性。TMB_4和LuH_6对塔崩磷酸化AChE有较强重活化,而对未老化梭曼膦酸化AChE无重活化,HI-6则相反,对未老化梭曼膦酰化AChE重活化效果好,而对塔崩磷酰化AChE无重活化作用。塔崩和梭曼膦酰化AChE在未老化以前对药物的响应就有所不同,毒剂残基的空间效应可能起重要作用。     

胆碱酯酶的基础与临床研究进展

简述红细胞乙酰胆碱酯酶（ＡＣｈＥ）和血浆丁酰胆碱酯酶（ＢｕＣｈＥ）的基础和临床应用研究的若干进展。红细胞ＡＣｈＥ活性测定已广泛应用于有机磷酸酯（ＯＰ）和氨基甲酸酯杀虫剂中毒的诊断以及职业接触的健康监护。血浆ＢｕＣｈＥ由肝细胞合成,是评价肝细胞合成功能的灵敏指标。急性重型肝炎、慢性活动性肝炎和肝硬化时血浆ＢｕＣｈＥ活性明显降低,可作为这些肝病的诊断标准之一。氨基甲酰化ＢｕＣｈＥ和有机磷酰化ＢｕＣｈＥ之间动力学特征的差别可用于这两类杀虫剂中毒的鉴别诊断：前者在酶抑制率≥４０％时,经时反应曲线呈非线性,而后者呈线性。     

Field verification of Test-mate ChE

The objective was to evaluate the ability of the Test-mate ChE to determine acetylcholinesterase (AChE) activity under field conditions. To mimic nerve agent exposure, the U.S. Army Medical Research Institute of Chemical Defense spiked blood samples with variable amounts of soman. Blinded to the identity of the samples, the 520th Theater Army Medical Laboratory tested the samples during a field training exercise inside their environmentally controlled mobile facility. The technicians repeated measurements for 6 consecutive days, and on 1 of the days repeated the measurements six times. The technicians accurately identified all of the samples and quantified the AChE activity. The major trend was that the Test-mate ChE is more precise and reproducible for smaller doses of soman. The results were reliable over all temperatures during the field exercise. In conclusion, the Test-mate ChE is a reliable field instrument to determine blood AChE activity.     

Synthesis and evaluation of radioiodine-labelled CP-118,954 for the in-vivo imaging of acetylcholinesterase

OBJECTIVES: Alzheimer's disease (AD) is characterized by reduced acetylcholinesterase (AChE) activity in the post-mortem tissues of AD patients. Therefore, AChE has been an attractive target for the diagnosis of AD. In the present study, 5,7-dihydro-3-[2-(1-(phenylmethyl)-4-piperidinyl)ethyl]-6H-pyrrolo[3,2-f]-1,2-benzisoxazol-6-one (CP-118,954), a potent AChE inhibitor, was labelled with radioiodine and evaluated as an AChE imaging agent for SPECT. METHODS: Radioiodine-labelled CP-118,954 was prepared from CP-144,885 and [(125)I]iodobenzyl bromide, and anti-AChE activities of iodine-substituted CP-118,954 were measured. Metabolism studies were carried out in samples of blood and whole brain of mice injected with 2-[(123)I]iodo-CP-118,954 ((123)I-1). Tissue distribution studies were also performed in mice injected with I-1, and samples of blood, thyroid, stomach, and brain tissue (cerebellum, striatum and cortex) were removed, weighed and counted. RESULTS: Of the ligands, 2-iodo-CP-118,954 exhibited higher binding affinity for AChE (IC50=24 nM) than the other positional isomers. 2-[(125)I]Iodo-CP-118,954 was found to have a lipophilicity (log P=2.1) favouring brain permeability and metabolic stability in mouse brain, but a marginal target (striatum) to non-target (cerebellum) uptake ratio (1.1) in mouse brain. CONCLUSION: This result demonstrates that 2-[(125)I]iodo-CP-118,954 may be unsuitable for AChE imaging. These findings suggest that radioligands suitable for AChE imaging should have not only a specific structure but also a sub-nanomolar to low nanomolar IC50.     

Simplified methods for in vivo measurement of acetylcholinesterase activity in rodent brain

Simplified methods for in vivo studies of acetylcholinesterase (AChE) activity in rodent brain were evaluated using N-[¹¹C]methylpiperidinyl propionate ([¹¹C]PMP) as an enzyme substrate. Regional mouse brain distributions were determined at 1 min (representing initial brain uptake) and 30 min (representing trapped product) after intravenous [¹¹C]PMP administration. Single time point tissue concentrations (percent injected dose/gram at 30 min), tissue concentration ratios (striatum/cerebellum and striatum/cortex ratios at 30 min), and regional tissue retention fractions (defined as percent injected dose 30 min/percent injected dose 1 min) were evaluated as measures of AChE enzymatic activity in mouse brain. Studies were carried out in control animals and after dosing with phenserine, a selective centrally active AChE inhibitor; neostigmine, a peripheral cholinesterase inhibitor; and a combination of the two drugs. In control and phenserine-treated animals, absolute tissue concentrations and regional retention fractions provide good measures of dose-dependent inhibition of brain AChE; tissue concentration ratios, however, provide erroneous conclusions. Peripheral inhibition of cholinesterases, which changes the blood pharmacokinetics of the radiotracer, diminishes the sensitivity of all measures to detect changes in central inhibition of the enzyme. We conclude that certain simple measures of AChE hydrolysis rates for [¹¹C]PMP are suitable for studies where alterations of the peripheral blood metabolism of the tracer are kept to a minimum.     

Synthesis and evaluation of 5,7-dihydro-3-[2-[1-(4-[18F]-fluorobenzyl)-4-piperidinyl]ethyl]-6H-pyrrolo[3,2-f]-1,2-benzisoxazol-6-one for in vivo mapping of acetylcholinesterase

OBJECTIVES: Acetylcholinesterase (AChE) is an important cholinergic marker for the diagnosis of Alzheimer's disease (AD). A recent study has demonstrated that C-labelled 5,7-dihydro-7-methyl-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-6H-pyrrolo[3,2-f]-1,2-benzisoxazol-6-one (CP-126,998) shows promising results. The demethylated form of this ligand (CP-118,954) is a more potent and selective inhibitor than CP-126,998. In this study, therefore, CP-118,954 was labelled with F and evaluated for the in vivo mapping of AChE. METHODS: The 4-fluoro (1). and 2-fluoro (2). derivatives of CP-118,954 were synthesized from 4-methyl-3-nitroanisole in 11 steps. Their in vitro binding affinities to AChE were measured using Ellman's method. The preparation of [F]-1 was carried out by reductive alkylation of the piperidine precursor with 4-[F]-fluorobenzaldehyde, followed by high-performance liquid chromatography (HPLC) purification. In vitro autoradiography was performed by incubating rat brain coronal slices with [F]-1. Tissue distribution studies were performed in mouse brain and the data were expressed as the percentage of the injected dose per gram of tissue (%ID x g). RESULTS: Two fluorine-substituted AChE inhibitors were synthesized and their in vitro binding data showed that the 4-fluoro and 2-fluoro derivatives (1 and 2) had similar or superior binding affinity to that of the unsubstituted ligand, CP-118,954. The F-labelled ligand was synthesized in 20-35% radiochemical yield (EOS) and with high effective specific activity (36-42 GBq x micromol). Autoradiography showed high uptake of [F]-1 in the striatum and this striatal uptake was completely inhibited by the unlabelled ligand 1. Tissue distribution studies demonstrated that high radioactivity was accumulated in the striatum, an AChE-rich region. CONCLUSIONS: This study demonstrates that [F]-1 may hold promise as a radioligand for the in vivo mapping of AChE.     

Is subnanomolar binding affinity required for the in vivo imaging of acetylcholinesterase? Studies on 18F-labeled G379

Acetylcholinesterase (AChE) is an important cholinergic marker of Alzheimer's disease (AD) and shows reduced activity in postmortem AD brain tissues. 1-(4-Fluorobenzyl)-4-[(5,6-dimethoxy-1-oxoindan-2-fluoro-2-yl)methyl]piperidine (G379, ), an AChE inhibitor with a subnanomolar IC(50) (0.56 nM), was prepared as a (18)F-labeled radioligand ([(18)F]) and evaluated in mice. Metabolism studies of [(18)F] showed no metabolites in the mouse brain. Tissue distribution studies demonstrated its uniform regional distribution in the mouse brain, suggesting that this radioligand is not suitable for the in vivo imaging of AChE. This result along with reports on radiolabeled N-benzylpiperidine lactam benzisoxazole (IC(50) < 1 nM) and other radiolabeled benzylpiperidine derivatives (IC(50) > 1 nM) suggested that a subnanomolar IC(50) may not be the only important factor in determining the suitability of a radioligand for in vivo studies of AChE.     

Forensic significance of acetylcholine esterase histochemistry in organophosphate intoxication

Summary The reduction of acetylcholine esterase (AChE) activity or the complete blocking of AChE to be observed by histochemical demonstration of AChE in tissue after experimental and spontaneous (human) organophosphate intoxication (especially paraoxone = E 600 and parathion = E 605) should be interpreted as an indication of an in vivo inhibition of the cholinergic system. In animal experiments, a relationship was demonstrated between AChE activity and the applied dose of organophosphorous compounds. In addition, enzyme inhibition was observed in in vitro systems using AChE-containing mouse tissue sections pretreated with organophosphate solutions or with body fluids containing organophosphates. Examination of the concentration dependency indicated that the inhibiting solution must contain at least 0.15 g/ml paraoxone or 5 mg/ml parathion to block AChE in the section. Using the same in vitro system, a half-life of 6–7 min was established for the paraoxone inactivating enzyme in blood. The in vivo and in vitro inhibited AChE was reactivated by consecutive treatment of blocked sections with toxogonin. This possibility of reactivation therefore allows qualitative classifications of the AChE-inhibiting toxin to the alkylphosphates. The postmortem persistence of the AChE inhibitory effect was demonstrable for about a 2-month interval. Since the histochemically demonstrable activity of the enzyme AChE is more or less constant during a postmortem interval of at least 70h, the model of histochemical demonstration is a method which provides a morphological equivalent for acute organophosphate intoxication.Dedicated to Prof. Dr. J. Peiffer on occasion of his 60th birthday     

肟类重活化剂对抗氧化乐果抑制离体大鼠膈肌的研究

在离体大鼠膈神经－膈肌标本（膈ＮＭＰ）上观察了和的肟类重活化剂氯磷定碘磷定、双复磷和ＨＩ－６对抗氧化氯对标本的抑制作用，发现４种药物在对抗氧化乐果抑制离体大鼠膈ＮＭＰ的作用中均有对中毒酶的重活化作用，其强弱排序为：双复磷〉氯磷定、碘磷定〉ＨＩ－６。ＨＩ－６具有较强的直接生理对抗作用。