不同有机磷酸酯磷酰化乙酰胆碱酯酶活性中心的构象…

通过观察２倍肟化合物ＨＩ－６和ＨＧＧ－４２及它们的４位肟异构体对不同有机磷毒剂抑制的ＡＣｈＥ的重活化作用发现塔崩、梭曼、沙林等有机磷毒剂磷酰化的ＡＣｈＥ活性中心的构象可能存在着明显差异；又从变构剂Ｃ１０和丙吡啶对ＴＭＢ４重活化这几种毒剂磷酰化ＡＣｈＥ的影响中证实塔崩磷酰化ＡＣｈＥ活性中心构象与沙林、梭曼和ＶＸ３种毒剂磷酰化的ＡＣｈＥ明显不同。     

生化毒理学的研究——梭曼与乙酰胆碱酯酶作用的生化机理

梭曼(soman,特己基甲基氟膦酸酯)、沙林(sarin,异丙基甲基氟膦酸酯)、维埃克斯[VX,O-乙基-S-(2-二异丙胺乙基)甲基膦酸酯]等是一类重要的神经性化学战剂,其中以梭曼中毒最难防治。它们对乙酰胆碱酯酶(AChE)产生极强的抑制作用,使乙酰胆碱在神经末梢大量集聚,引起抽筋、惊厥,最后死亡。氯磷啶、双磷啶等重活化剂是神经性毒剂中毒急救中的重要药物,它们可以使沙林和VX抑制的AChE恢复活性,但对梭曼抑制的AChE很     

梭曼和沙林对电鳐电器官乙酰胆碱酯酶抑制作用动力学

梭曼和沙林是毒性很强的有机磷神经毒剂,它们的共同特点是对胆碱酯酶（ＡＣｈＥ,ＥＣ３．１．１．７）都有很强的抑制作用,但两者又不完全相同．过去人们对梭曼和沙林的毒性、毒理研究得较多,而对其对ＡＣｈＥ抑制作用动力学研究得较少,且相互间差异较大．为了对这一...     

杀虫药剂抗性家蝇品系乙酰胆碱酯酶基因的特征分析

乙酰胆碱酯酶(AChE)是有机磷和氨基甲酸酯类杀虫药剂的作用靶标,这两大类杀虫药剂的广泛应用导致了昆虫对抗性的选择。靶标的修饰是某些昆虫产生抗性的分于机理,这种抗性是和AChE的变更型相关的,这些变更型的酶显示出对杀虫药剂的不被感性。利用RT-PCR和Streptavidin偶联磁珠技术从两种抗性家蝇(Musca domestica)品系D3和Kash中分别分离了AChE基因并测定了其按苷酸颅序。eDNA的可读框长2082bp．由此推导出了AChE的氨基酸顺序,通过与敏感家蝇品系Cooper的比较,发现了一些核苷酸顺序差异和4个氨基酸点突变,其中3个替代可能与杀虫药剂不敏感性有关。这一结果表明D3和Kash均属于CH₂抗性类型。     

3种肟类化合物对中毒乙酰胆碱酯酶的重活化能力比较

3种肟类化合物(TMB4、LUH6和2-PAM-Cl)对辛硫磷抑制的鲫鱼、蟑螂和家蝇的乙酰胆碱酯酶(AChE)均具有重活化作用。初步比较研究了3种肟类化合物对中毒AChE的重活化能力,发现它们对被抑制的蟑螂和家蝇的AChE重活化能力差异不显著;但是对被抑制的鲫鱼的AChE来说,TMB4和L H6的重活化能力明显比2-PAM-Cl强。推测这3种肟类化合物对蟑螂、家蝇和鲫鱼的中毒AChE的活化能力的差异可能是由脊椎动物与无脊椎动物AChE本身的差异性造成的。     

Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用

为探讨Smac/DIABLO在过氧化氢(H₂O₂）所致C₂C₁₂肌原细胞凋亡中的作用,采用Hoechst 33258染色,观察H₂O₂ (0.5 mmol/L)处理C₂C₁₂肌原细胞不同时间后,细胞核形态学改变并计算凋亡核百分率,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带,利用细胞成分分离后蛋白质印迹分析H₂O₂是否导致Smac/DIABLO从线粒体释放,采用Caspase检测试剂盒及蛋白质印迹分析Caspase-3和Caspase-9的活化,转染Smac/DIABLO基因,观察Smac/DIABLO过表达对H₂O₂所致的C₂C₁₂肌原细胞凋亡的影响.结果表明:H₂O₂处理1 h后,Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放入胞浆,2 h更明显;H₂O₂处理4 h后,Caspase-3和Caspase-9活化,12 h达高峰;H₂O₂处理24 h后,C₂C₁₂肌原细胞显示特征性的凋亡形态改变,凋亡核百分率明显升高,DNA电泳出现明显“梯状”条带.与单纯过氧化氢损伤组相比,Smac/DIABLO高表达的C₂C₁₂肌原细胞经过氧化氢损伤组的Caspase-3和Caspase-9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显.结果表明,H₂O₂可导致Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放,促进Caspase-9和Caspase-3的活化而促进细胞凋亡的发生.     

螺旋藻POD、CAT和SOD同工酶的研究*

采用聚丙烯酰胺凝胶电泳法对毛乌素沙地碱湖的钝顶螺旋藻S3和鄂尔多斯螺旋藻S4与国外引进的钝顶螺旋藻S₁和极大螺旋藻S₂ 的POD、CAT和SOD同工酶进行了比较研究。结果表明 :4个样品的 3种酶同工酶带数目不同 ,依次是S₄ >S₃>S₁>S₂ ;S₃和S₄ 酶带数多 ,对环境适应性强 ,进化程度较高。螺旋藻不同种间的酶谱相似系数     

竹红菌乙素敏化的人红细胞膜结构光损伤的Raman光谱特征

采用Raman光谱从分子水平揭示了竹红菌乙素光敏损伤的人红细胞膜发生膜蛋白交联和膜脂脂质过氧化导致其功能变化 ;膜流动性和离子通透性增加的本质是竹红菌乙素产生的活性氧 ( ¹O₂ ,O₂ ^-·和·OH等 )破坏了红细胞膜的有序结构 ,使膜蛋白主链结构的α 螺旋、β 折叠明显减少 ,无规卷曲增加并使其侧链结构的巯基基团、吲哚环、对羟苯基环、单基取代苯基环等也明显减少 .与此同时 ,随着光照时间的增加 ,膜脂的反式构象呈先增加后减少的趋势 ,它的扭曲构象则正好相反 .膜蛋白和膜脂构象不灵敏的CH₂ 和CH₃弯曲振动谱线的明显下降 ,揭示它们有链的断裂 .     

抗菌肽的固相合成、分离纯化与构效关系的研究

应用手工固相DCC/HOBt法合成Cecropin B、shival、ABP 3、WHD4种抗菌肽。各抗菌肢的C端均为酰胺化。最终用}珏,LC分离纯化。这4种肽对大肠杆菌K₁₂D₃₁野生型大肠杆菌、产气杆菌及4种植物病原菌青枯菌、临床致病型大肠杆菌、伤寒杆菌、硝酸盐杆菌均有明显的杀伤或抑制作用．对白血病细胞K 560、肺癌细胞A 549也有杀伤作用。其中尤以wHD杀伤作用最为明显,并用对其它3种肽不能作用的枯草杆菌、绿脓杆菌也略有抑制。而shiva l对细菌的作用较其它3种肽弱,另外还用cD谱对4种抗菌肽的溶液构象做了初步研究。     

氮素形态对杉木幼苗侧根生长和叶片光合特性的影响

以3月龄的杉木实生苗为试验材料,分析了不同氮素形态——硝态氮(NO₃^- N)、铵态氮(NH4⁺ N)和硝酸铵(NH₄NO₃)(氮素浓度均为3 mmol·L^-1)对杉木幼苗侧根生长、叶片光合气体交换参数和叶绿素荧光参数的影响,以揭示杉木幼苗对不同形态氮的偏好性,以及不同形态氮肥下杉木幼苗侧根生长和光合生理的响应特征,为杉木苗期氮肥管理提供理论依据。结果显示：(1)不同氮素形态对杉木幼苗地上部和侧根生物量具有显著影响,其中NH₄⁺ N处理下幼苗地上部和侧根生物量最大,NO₃^- N处理次之,而NH₄NO₃处理最小。(2)NH₄⁺ N和NO₃^- N处理下杉木幼苗总根长、根系总表面积和根系总体积均显著高于NH₄NO₃处理(P＜0.05),且NH₄⁺ N处理又显著高于NO₃^- N处理,但不同氮形态处理间侧根数量差异不显著。(3)NH₄⁺ N处理下杉木幼苗叶片净光合速率、气孔导度和蒸腾速率明显高于NO₃^- N和NH₄NO₃处理,但NO₃^- N和NH₄NO₃处理之间无明显差异。(4)NH₄⁺ N处理下杉木叶片初始荧光强度低于NO₃^- N处理,而最大荧光强度、可变荧光强度和PSⅡ潜在活性却高于全硝氮和硝铵氮处理。上述结果表明,NH₄⁺ N处理不仅有利于杉木幼苗侧根生长发育,且其叶片具有较强的光合能力,较高的PSⅡ中心稳定性、光化学活性以及电子传递效率,从而更有利于植株生长。因此,从根系生长和光合特性来看,杉木幼苗对铵态氮具有偏好性。     

New bispyridinium oximes: in vitro and in vivo evaluation of their biological efficiency in soman and tabun poisoning

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still a challenge for the scientific community. This study investigated the interactions of four bispyridinium oximes with human erythrocyte acetylcholinesterase (AChE) and their effects on soman- and tabun-poisoned mice. Oximes HI-6 and TMB-4 were used for comparison. These oximes inhibited AchE with inhibitory potency (IC(50)) ranging from 0.02 to 1.0 mM. The best reactivating potency (%R) was obtained with K074, when AChE was inhibited by tabun. The protective potency (P(50)) of all oximes in human erythrocyte AChE inhibited by soman and tabun could not be determined. In tabun-poisoned mice very good antidotal efficacy was obtained with K027, K048, and K074, which makes them interesting for future investigation. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.     

New bispyridinium oximes: In vitro and in vivo evaluation of their biological efficiency in soman and tabun poisoning

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still a challenge for the scientific community. This study investigated the interactions of four bispyridinium oximes with human erythrocyte acetylcholinesterase (AChE) and their effects on soman- and tabun-poisoned mice. Oximes HI-6 and TMB-4 were used for comparison. These oximes inhibited AchE with inhibitory potency (IC₅₀) ranging from 0.02 to 1.0 mM. The best reactivating potency (%R) was obtained with K074, when AChE was inhibited by tabun. The protective potency (P₅₀) of all oximes in human erythrocyte AChE inhibited by soman and tabun could not be determined. In tabun-poisoned mice very good antidotal efficacy was obtained with K027, K048, and K074, which makes them interesting for future investigation. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.     

Inhibitors of Renin as Potential Therapeutic Agents

Abstract

The reaction of usual (U) and atypical (A) cholinesterase phenotypes was studied with six organophosphorus compounds, two pyridinium oximes (HI-6 and PAM-2) and with 4–4-bipyridine (4,4-BP). No difference in the inhibition rate constants for the two phenotypes was found with the progressive inhibitors tabun, sarin, paraoxon and soman. The other two progressive inhibitors, VX and the positively charged phosphostigmine, inhibited the U phenotype more strongly than the A phenotype.

The positively charged reversible inhibitor HI-6 showed a higher affinity for the U than for the A phenotype, while PAM-2 and the non-charged 4,4′-BP did not show a significant difference in their affinity towards the two enzymes.

Both phenotypes phosphylated by VX or sarin were reactivatable by HI-6 and PAM-2, and the A phenotype was always reactivated more slowly than the U phenotype. The paraoxon-inhibited phenotypes were reactivated at equal rates with PAM-2 but were not reactivated with HI-6. The phosphylated phenotypes did not reactivate spontaneously during one hour.

The effect of reversible inhibitors upon the rate of phosphylation (protection) was tested with HI-6 (for inhibition by soman, tabun and paraoxon) and with 4,4′-BP (for inhibition by soman). By applying the concentrations of the protectors equal to their enzyme/inhibitor dissociation constants, a better protection of the U than of the A phenotype was achieved by HI-6, but equal protection was given by 4,4′-BP.     

A comparison of the therapeutic and reactivating efficacy of newly developed bispyridinium compounds (K206, K269) with currently available oximes against tabun in rats and mice

The potency of newly developed bispyridinium compounds (K206, K269) in reactivating tabun-inhibited acetylcholinesterase and eliminating tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies which determined percentage of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats showed that the reactivating efficacy of both newly developed oximes is comparable with obidoxime and trimedoxime in blood but lower than the reactivating potency of trimedoxime and obidoxime in the diaphragm and brain. Nevertheless, the differences in reactivating efficacy of obidoxime, trimedoxime and K206 was not significant while the potency of K269 to reactivate tabun-inhibited acetylcholinesterase was significantly lower. Both newly developed oximes were also found to be relatively efficacious in elimination of the lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy corresponds to the therapeutic potency of obidoxime. The oxime HI-6, relatively efficacious against soman, did not seem to be an adequately effective oxime in reactivation of tabun-inhibited AChE and to counteract lethal effects of tabun. Both newly developed oximes (K206, K269) are significantly more efficacious in reactivating tabun-inhibited AChE in rats and to eliminate lethal toxic effects of tabun in mice than the oxime HI-6 but their reactivating and therapeutic potency does not prevail over the effectiveness of currently available obidoxime and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.     

A comparison of the therapeutic and reactivating efficacy of newly developed bispyridinium compounds (K206, K269) with currently available oximes against tabun in rats and mice

The potency of newly developed bispyridinium compounds (K206, K269) in reactivating tabun-inhibited acetylcholinesterase and eliminating tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies which determined percentage of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats showed that the reactivating efficacy of both newly developed oximes is comparable with obidoxime and trimedoxime in blood but lower than the reactivating potency of trimedoxime and obidoxime in the diaphragm and brain. Nevertheless, the differences in reactivating efficacy of obidoxime, trimedoxime and K206 was not significant while the potency of K269 to reactivate tabun-inhibited acetylcholinesterase was significantly lower. Both newly developed oximes were also found to be relatively efficacious in elimination of the lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy corresponds to the therapeutic potency of obidoxime. The oxime HI-6, relatively efficacious against soman, did not seem to be an adequately effective oxime in reactivation of tabun-inhibited AChE and to counteract lethal effects of tabun. Both newly developed oximes (K206, K269) are significantly more efficacious in reactivating tabun-inhibited AChE in rats and to eliminate lethal toxic effects of tabun in mice than the oxime HI-6 but their reactivating and therapeutic potency does not prevail over the effectiveness of currently available obidoxime and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.     

The role of the oximes HI-6 and HS-6 inside human acetylcholinesterase inhibited with nerve agents: a computational study

The oximes 4-carbamoyl-1-[({2-[(E)-(hydroxyimino) methyl] pyridinium-1-yl} methoxy) methyl] pyridinium (known as HI-6) and 3-carbamoyl-1-[({2-[(E)-(hydroxyimino) methyl] pyridinium-1-yl} methoxy) methyl] pyridinium (known as HS-6) are isomers differing from each other only by the position of the carbamoyl group on the pyridine ring. However, this slight difference was verified to be responsible for big differences in the percentual of reactivation of acetylcholinesterase (AChE) inhibited by the nerve agents tabun, sarin, cyclosarin, and VX. In order to try to find out the reason for this, a computational study involving molecular docking, molecular dynamics, and binding energies calculations, was performed on the binding modes of HI-6 and HS-6 on human AChE (HssAChE) inhibited by those nerve agents.     

A comparison of reactivating efficacy of newly developed oximes (K074, K075) and currently available oximes (obidoxime, HI-6) in soman, cyclosarin and tabun-poisoned rats

The potency of newly developed oximes (K074, K075) and commonly used oximes (obidoxime, HI-6) to reactivate nerve agent-inhibited acetylcholinesterase was evaluated in rats poisoned with soman, tabun or cyclosarin at a lethal dose corresponding to their LD(50) value. In vivo determined percentage of reactivation of soman-inhibited blood and brain acetylcholinesterase in poisoned rats showed that only the oxime HI-6 was able to reactivate soman-inhibited acetylcholinesterase in the peripheral (blood) as well as central (brain) compartment. In vivo determined percentage of reactivation of tabun-inhibited blood and brain acetylcholinesterase in poisoned rats showed that obidoxime is the most efficacious reactivator of tabun-inhibited acetylcholinesterase among studied oximes in the peripheral compartment (blood) while K074 seems to be the most efficacious reactivator of tabun-inhibited acetylcholinesterase among studied oximes in the central compartment (brain). In vivo determined percentage of reactivation of cyclosarin-inhibited blood and brain acetylcholinesterase in poisoned rats showed that HI-6 is the most efficacious reactivator of cyclosarin-inhibited acetylcholinesterase among studied oximes. Due to their reactivating effects, both newly developed K oximes can be considered to be promising oximes for the antidotal treatment of acute tabun poisonings while the oxime HI-6 is still the most promising oxime for the treatment of acute soman and cyclosarin poisonings.     

New quaternary pyridine aldoximes as casual antidotes against nerve agents intoxications

In this work, the ability of four newly synthesized oximes--K005 (1,3-bis(2-hydroxyiminomethylpyridinium) propane dibromide), K027 (1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide), K033 (1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide) and K048 (1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide) to reactivate acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by nerve agents is summarized. Reactivation potency of these compounds was tested using standard in vitro reactivation test. Tabun, sarin, cyclosarin and VX agent were used as appropriate testing nerve agents. Rat brain AChE was used as a source of the enzyme. Efficacies of new reactivators to reactivate tabun-, sarin-, cyclosarin- and VX-inhibited AChE were compared with the currently used AChE reactivators (pralidoxime, obidoxime and HI-6). Oxime K048 seems to be promising reactivator of tabun-inhibited AChE. Its reactivation potency is significantly higher than that of HI-6 and pralidoxime and comparable with the potency of obidoxime. The best reactivator of sarin-inhibited AChE seems to be oxime HI-6. None of the new AChE reactivators reached comparable reactivation potency. The same results were obtained for cyclosarin-inhibited AChE. However, oxime K033 is also potent reactivator of AChE inhibited by this nerve agent. In the case of VX inhibition, obidoxime and new oximes K027 and K048 seem to be the best AChE reactivators. None from the currently tested AChE reactivators is able to reactivate AChE inhibited by all nerve agents used and, therefore, the search for new potential broad spectrum AChE reactivators is needed.     

Oximes: Reactivators of phosphorylated acetylcholinesterase and antidotes in therapy against tabun poisoning

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime, found as the most effective reactivators, have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE. Three of our tested pyridinium oximes K027, K048, K074, along with TMB-4, were the most promising for AChE reactivation. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Herein, we showed that a promising treatment in tabun poisoning by selected oximes and atropine could be improved if oximes are also used in pretreatment. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.     

A comparison of the ability of a new bispyridinium oxime--1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide and currently used oximes to reactivate nerve agent-inhibited rat brain acetylcholinesterase by in vitro methods

The efficacy of a new bispyridinium oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide, called K048, and currently used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. The new oxime K048 was found to be a more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than pralidoxime (in the case of VX, tabun and cyclosarin), obidoxime (cyclosarin and tabun) and HI-6 (tabun) but it did not reach the efficacy of currently used oximes for the reactivation of acetylcholinesterase inhibited by sarin. Thus, the oxime K048 seems to be a relatively efficacious broad spectrum acetylcholinesterase reactivator and, therefore, it could be useful for the treatment of a nerve agent-exposed population if information about detection of the type of nerve agent is not available.