AMPA受体和相关蛋白在束缚应激大鼠相关脑区的表达变化及逍遥散对其影响

目的：观察海马及杏仁核α-氨基羟甲基恶唑丙酸（AMPA）受体亚基和相关调节蛋白在束缚应激状态下蛋白表达变化及逍遥散的调节作用。方法：使用每天捆绑3 h的方法制作慢性束缚应激动物模型,并用逍遥散进行干预,分别于7 d后和21 d后用Western blot方法检测各组大鼠海马CA1区、CA3区、齿状回（DG）和杏仁核的AMPA受体亚基GluR2/3及N-乙基顺丁烯二酰亚胺敏感性的融合蛋白（NSF）、PKC作用蛋白1（PICK1）蛋白表达的情况。结果：7 d应激可使DG和杏仁核的GluR2/3、NSF表达显著降低（P均〈0.05）,使PICK1在CA1区的表达量显著增多（P〈0.05）,逍遥散对PICK1变化显示出一定调节作用。21 d应激可使CA1区的GluR2/3、NSF表达升高,其中GluR2/3有显著性差异（P〈0.01）,而在杏仁核表达有降低趋势,逍遥散对其均有显著调节作用（均为P〈0.05）,21 d应激使杏仁核PICK1表达量出现升高趋势,逍遥散可显著降低其表达（P〈0.05）。结论：AMPA受体在短期重复应激和慢性应激状态下反应不同,海马和杏仁核反应相反,逍遥散对慢性应激状态下AMPA受体表达的调节作用较短期重复应激强。     

Deriving Toxicity Values for Organophosphate Nerve Agents: A Position Paper in Support of the Procedures and Rationale for Deriving Oral RfDs for Chemical Warfare Nerve Agents

During the process of deriving oral Reference Dose (RfDs) values for chemical warfare agents, several issues arose regarding the identification of adverse effect levels and the application of uncertainty factors. For those agents that function as cholinesterase inhibitors (e.g., agents VX, GA, GB, and GD), these issues included the following: (1) Is the endpoint of blood cholinesterase inhibition an indicator of toxicity or a biomarker of exposure? (2) Can an experimental animal species be more sensitive than humans, thereby eliminating the need for an animal-to-human uncertainty factor? (3) Can the uncertainty factor that is used to extrapolate from a lowest-observed adverse-effect-level (LOAEL) to a no-observed-adverse-effect-level (NOAEL) be less than the default value of 10? (4) Can an oral RfD be derived from non-oral toxicity data? (5) Can an uncertainty factor of less than 10 be used to extrapolate from subchronic to chronic exposure (e.g., is the critical effect adequately described by the subchronic exposure data)? (6) What constitutes an adequate data base for organophosphate cholinesterase inhibitors, and what uncertainty factor should be used for an incomplete data base? Analysis of relevant data resulted in the following selection and justifications of uncertainty factors. For uncertainty associated with intraspecies extrapolation (UF_H), physiologic and pathologic conditions affecting cholinest-erase activity levels justified maintaining a UF_H of 10 for all of the nerve agents. Because available data indicated that humans tended to be more sensitive than rats regarding anticholinesterase effects, an interspecies variability (UF_A) factor of 10 was retained for agents GA, GB, and GD. For agent VX, however, the available data revealed that the domestic sheep test species exhibited sensitivity equivalent to or greater than that of humans thereby justifying a UF_A of 1. For uncertainties regarding extrapolation from subchronic-to- chronic exposure data, consideration of information on the physiology of cholinergic systems and the available toxicity data for the nerve agents and other cholinest-erase inhibitors indicated that a UF_S of 3 was justified for all four of the nerve agents. For uncertainties regarding LOAEL-to- NOAEL extrapolation (UF_L), the selection of agent GB, GD, and VX doses resulting in cholinesterase inhibition in the absence of clinical signs of toxicity (biomarker of exposure) justified this endpoint as a minimal LOAEL and a UF_L of 3. For agent GA, a NOAEL was used, and therefore no UF_L was required. The uncertainty factor for data base completeness (UF_D), was based upon several considerations. Of primary concern was the fact that chronic toxicity studies are not considered an essential component of the data base requirements for cholinesterase inhibitors because of the unlikelihood that the endpoint will change with an increase in exposure time beyond that defined as a subchronic exposure. Additionally, limited data regarding reproductive and developmental toxicity were not considered to represent critical toxicity endpoints for the nerve agents or cholinesterase inhibitors in general. Although the data base for agents GA, GB, and GD were lacking reproductive and developmental toxicity data to some extent, a UF_D of 3 was justified for the aforementioned reasons. The data base for agent VX was considered complete and a UF_D of 1 was selected for development of the RfD for this agent. A modifying factor (MF) to reflect qualitative assessment of additional uncertainties in the critical study or data base that are not addressed by uncertainty factors was limited to agent GA due to the route-to-route (i.e., intraperitoneal to oral) extrapolation and to insure the equivalent oral NOAEL was not overestimated. This article provides a brief overview of the nerve agents, information on cholinergic systems that is pertinent to deriving toxicity values for nerve agents and other organophosphate cholinesterase inhibitors, and a discussion of key issues regarding the use of uncertainty factors in RfD derivations.     

MgCl2-sensitive and Gpp(NH)p-sensitive antagonist binding states of rat heart muscarinic receptors: preferential detection at ambient temperature assay and location in two subcellular fractions

Summary Some novel observations dealing with antagonist binding to cardiac particulate muscarinic receptors are described. Gpp(NH)p increased (2–3 fold) the specific binding of [³H]-QNB or [³H]-NMS, both potent muscarinic antagonists, to washed particles (WP), but not microsomes (MIC), when the binding was conducted at 30°C. Magnesium, on the other hand, increased (2–3 fold) the binding of these antagonists to MIC, but not to WP, under the same condition. The treatment of subcellular fractions with 0.2 mM N-ethylmaleimide (NEM), a sulfhydryl reagent, failed to significantly modify the respective stimulatory actions of either Gpp(NH)p on WP binding or of magnesium on MIC binding of these antagonists; treatment with dithiothreitol (1 mM) was also ineffective in this regard. Gpp(NH)p decreased K_d (WP) while magnesium increased K_d (MIC) for [³H]-QNB. Repeated freezing/thawing of isolated subcellular fractions abolished the stimulatory effect of magnesium on onist binding to MIC but not of Gpp(NH)p on WP antagonist binding; the freeze/thaw procedure per se increased MIC binding but not WP binding of these antagonists. When the binding was conducted at 4°C (24 hr), the stimulatory effect of Gpp(NH)p on [³H]-QNB binding was enhanced (6-fold) in the case of WP and was detectable (80%) in the case of MIC. Under this condition, the stimulatory effect of magnesium on [³H]-QNB binding was also enhanced (5-fold) in the case of MIC and became evident (200%) in the case of WP. The results of this work support the following views: (a) antagonist-occupied cardiac muscarinic receptors are capable of interaction with guanine nucleotide binding proteins (G protein like G₁,G_o) and such interaction influences antagonist binding properties (e.g. increased affinity) of the cardiac membrane-associated muscarinic receptors (b) magnesium influences (decreased affinity) antagonist binding properties by interacting with multiple sites of which some are likely associated with components other than G proteins of the particulate fractions (c) a pool of NEM-sensitive sulfhydryls involved in the regulation of Gpp(NH)p-sensitive agonist binding to cardiac muscarinic receptors is not involved in the regulation by either Gpp(NH)p or magnesium of antagonist binding in these subcellular fractions and (d) membrane fluidity and microenvironment surrounding the receptor and G proteins contribute to the actions of Gpp(NH)p and magnesium on antagonist binding.     

Induction of stress proteins in anoxic and hyperthermicSpodoptera frugiperda cells

In this study, we compare stress protein induction in anoxic and hyperthermicSpodoptera frugiperda cells. Anoxia transiently induces a cluster of heat shock proteins at 71 and 72 kDa. This is a subset of a larger group of stress proteins induced by heat shock. Several heat shock proteins reported in this study were previously undetected inS. frugiperda. With these additional proteins, the stress response of hyperthermicS. frugiperda closely resembles that ofDrosophila melanogaster. Prior investigations of stress protein induction during oxygen deprivation focused on mammalian cells. In sharp contrast to these cells, anoxicS. frugiperda cells neither induce glucose-regulated proteins nor suppress the heat shock family of 71/72 kDa proteins. These findings provide insight into the virtually unexplored area of stress protein induction in anoxic insect cells. In addition, they help to explain the effects of oxygen deprivation on heterologous protein yield from virally infected insect cells and to develop an oxygenregulated promoter for stably transformed insect cells.Abbreviations DO dissolved oxygen concentration - GRP's glucose-regulated proteins - HSP's heat shock proteins - ORP's oxygen-regulated proteins - PAGE polyacrylamide gel electrophoresis - Sf9 Spodoptera frugiperda cells     

不同性别及性腺功能对梭曼引起大鼠低温的影响

目的：研究不同性别及性腺功能对梭曼引起大鼠低温的影响。方法：用数字体温计测量大鼠的结肠温度,观察梭曼引起正常雄性和雌性大鼠低温反应的性别差异以及切除性腺后对其作用的影响。结果：①雌性大鼠对梭曼引起的低温反应比雄鼠更敏感。②切除雄性大鼠睾丸后能明显提高对梭曼低温反应的敏感性,而切除卵巢的雌性大鼠对梭曼的低温反应与模拟手术组无明显差异。结论：雄性和雌性大鼠对梭曼敏感性的性别差异主要取决于睾丸的功能。     

Synthesis and biological evaluation of some thiazole derivatives as new cholinesterase inhibitors

In the present study, some thiazole derivatives were synthesized via the ring closure reaction of 1-[2-(2-oxobenzo[d]thiazol-3(2H)-yl)acetyl]thiosemicarbazide with various phenacyl bromides. The chemical structures of the compounds were elucidated by ¹H NMR, ¹³C NMR and mass spectral data and elemental analyses. Each derivative was evaluated for its ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) using a modification of Ellman’s spectrophotometric method. The compounds were also investigated for their cytotoxic properties using MTT assay. The most potent AChE inhibitor was found as compound 4e (IC₅₀?=?25.5?±?2.12 µg/mL) followed by compounds 4i (IC₅₀?=?38.50?±?2.12 µg/mL), 4c (IC₅₀?=?58.42?±?3.14 µg/mL) and 4g (IC₅₀?=?68?±?2.12 µg/mL) when compared with eserine (IC₅₀?=?0.025?±?0.01 µg/mL). Effective compounds on AChE exhibited weak inhibition on BuChE (IC₅₀ > 80 µg/mL). MTT assay indicated that the cytotoxic dose (IC₅₀?=?71.67?±?7.63 µg/mL) of compound 4e was higher than its effective dose.     

Changes of rat plasma total low molecular weight antioxidant level after tabun exposure and consequent treatment by acetylcholinesterase reactivators

These experiments were performed on a rat model. The rats were divided into eight groups and consequently exposed to either a saline solution (control), atropine or a combination of atropine and tabun. The reactivation efficacy of the oximes was estimated on the rats exposed to tabun, atropine and a reactivator of AChE. The oximes HI-6, obidoxime, trimedoxime, K203 and KR-22836 were used as representative compounds of commonly available and new AChE reactivators. Besides the positive effect of the administered reactivators on blood AChE activity, the sizable modulation of low molecular weight antioxidant (LMWA) levels was also determined. The LMWA levels in the the animals treated with the oxime reactivators were decreased in comparison with the animals treated by atropine alone. It was found that the levels of LMWA returned to the level found in the control animals when either trimedoxime, K203 or KR-22836 were administered. The principle of oxime reactivator function and a novel insight into AChE activity regulation and oxidative stress is discussed.