眼镜蛇属神经毒素研究概况

正世界范围内眼镜蛇属约28种[1]。我国有舟山眼镜蛇(naja atra)和孟加拉眼镜蛇(naja kaouthia)两种,前者过去又称为中华眼镜蛇[2]。眼镜蛇是腺管牙类毒蛇,其毒腺分泌的毒液是以神经毒为主的混合毒,即神经毒和血循毒。眼镜蛇毒神经毒素(cobra neurotoxin,CNT)是其毒液中毒性最大的成分,它能使动物产生呼吸抑制和骨骼肌麻痹,甚至死亡[3]。随着分子生物学和蛋白质组学的发展,眼镜蛇毒神经毒素的一些组分已得到分离、纯化、测定,并广泛应用于科研和临床。本文就眼镜蛇毒组分中神经毒素的研究与应用进     

肉毒神经毒素轻链的结构与功能

肉毒神经毒素(BoNT)是由肉毒梭菌产生的一类外毒素,它是目前自然界所发现的生物毒素中毒性最强的物质。近年来,BoNT制剂在临床治疗上呈现出广阔的应用前景。具有生物活性的BoNT由50kD的轻链(LC)和100kD的重链(HC)组成双链结构：LC具有锌内肽酶活性;HC为细胞结合和转位结构域。本文综述了BoNT的LC在一级结构、高级结构与功能的关系方面研究的新进展。     

蝮蛇毒和眼镜蛇毒对全血化学发光的影响

目的研究蝮蛇毒和眼镜蛇毒对吞噬细胞的免疫抑制作用。方法以氢化可的松为阳性对照物,用化学发光法测定样品清除非细胞体系产生的活性氧,以及样品对酵母多糖诱导的全血化学发光的抑制作用。结果蝮蛇毒和眼镜蛇毒对非细胞体系产生的Ｏ·２和Ｈ２Ｏ２的清除作用都较弱;对全血化学发光有抑制作用,特别是酵母多糖起刺激作用之前加入蛇毒的抑制作用强于酵母多糖起作用之后加入蛇毒的抑制作用。这提示,这两种蛇毒使吞噬细胞的吞噬免疫功能下降,造成吞噬细胞的呼吸爆发减弱,导致活性氧产生不足,化学发光强度下降。眼镜蛇毒的这种免疫抑制作用比氢化可的松还强。结论蝮蛇毒和眼镜蛇毒具有氢化可的松样的免疫抑制作用,其中眼镜蛇毒可能具有作为临床免疫抑制剂使用的价值。     

《动物学研究》增刊简介

《动物学研究》第2卷第4期增刊(1981) 本期增刊为“蛇毒研究与蛇伤治疗”。主要内容是:福建产圆斑蝰蛇(Vipera russclli siamensis)蛇毒磷酯酶A的分离纯化及部分性质;毒性磷酯酶A_2的氨基酸组成和N-末端部分氨基酸顺序测定;浙江蝮蛇毒碱性磷脂酶A的分离纯化及性质;我国10种蛇毒磷脂酶A活力比较;广东金环蛇细胞毒素Ⅷ的化学组成和部分一级结构;眼镜蛇膜毒素对菠菜叶绿体光合膜的影响;中华眼镜蛇毒膜毒素对鼠肝线粒体的作用;眼镜王蛇毒中L-氨基酸氧化酶的分离纯     

紫外线^60Co照射对蛇毒毒性的影响

选择体重20g 左右小白鼠分别使用眼镜蛇毒、蝮蛇毒以及经过紫外线、~(60)Co 照射灭菌后的这两种蛇毒进行半数致死量的测定,结果经紫外线照射后的眼镜蛇毒和白眉蝮蛇毒的毒性降低了6.98%和6.41%.经~(60)Co 照射的眼镜蛇毒和蝮蛇毒的毒性降低了21%和33%.     

中国境内4种常见蛇毒的蛋白质组学研究

目的研究中华眼镜蛇毒、尖吻蝮蛇毒、银环蛇毒和金环蛇毒的蛋白构成。方法采用鸟枪蛋白质组学策略结合无标记定量方法检测4种蛇毒的蛋白种类及丰度。结果金属蛋白酶Aculysin-2、金属蛋白酶前体H_4、心脏毒素1e、磷脂酶B 81b、Thaicobrin、乙酰胆碱酯酶在多种毒液中均被检出,可能成为影响免疫鉴定方法准确度的因素。结论揭示不同蛇毒的蛋白构成的异同可以为蛇伤鉴定方法的开发与蛇伤救治提供基础。     

眼镜蛇毒心脏毒素的毒理及机制

眼镜蛇毒中含丰富的心脏毒素(Cardiotoxin,简称CTx),已分离提纯近60种,每种蛇毒中含2—4种CTx。1984年杜雨苍从广东产中华眼镜蛇毒(Naja Naja atra)中分离出该毒的第五种CTx,并命名为“细胞膜毒素     

广西眼镜蛇毒细胞毒素-2的分离纯化及其对大鼠肝星状细胞HSC-T6的作用

目的通过层析法从广西眼镜蛇毒中分离得到电泳纯的细胞毒素-2(CTX-2),探索CTX-2对大鼠肝星状细胞(HSC-T6)的增殖抑制作用。方法采用DEAE-Sepharose CL-6B阴离子交换层析、Spehadex G-50凝胶层析和Macro-prep High S阳离子交换层析结合的方法分离眼镜蛇毒粗毒;经SDS-PAGE电泳鉴定蛋白纯度;NanoLC-ESI-MS/MS质谱方法鉴定其组分并测定分子量;CCK-8法检测CTX-2对HSC-T6的增殖抑制作用,确定其最小有效浓度。结果眼镜蛇毒粗毒经分离纯化获得电泳纯的CTX-2,其分子量约为9.548 kD;不同浓度CTX-2作用于HSC-T6细胞24 h后,其增殖抑制作用随浓度增加而增大,呈量效关系,抑制增殖的最小有效浓度为8 mg/L,IC_(50)为11.52 mg/L。结论广西眼镜蛇毒粗毒经三步分离法得到电泳纯且具有高生物活性的CTX-2;广西眼镜蛇毒CTX-2可抑制HSC-T6细胞增殖,抑制作用呈量效关系。     

白介素-2的结构-功能关系

对白介素-2的一级结构和空间结构与其生物活性的关系,尤其是与白介素-2受体中不同亚基亲和力的关系作了概述和总结,对研制具有不同生物学活性的新型白细胞介素-2有重要的指导意义。     

蓝色染料配基亲和纯化眼镜蛇心脏毒素的研究

探索了蓝色染料（Cibacron Blue F3G-A）亲和分离中华眼镜蛇心脏毒素的可能性。采用环氧基活化法制备蓝色染料亲和介质,中性条件下提取眼镜蛇粗毒中的心脏毒素。Tricine系统SDS-PAGE多肽电泳和Lowry法蛋白定量分析纯化效果,发现蓝色染料琼脂糖一步纯化中华眼镜蛇心脏毒素的纯度达到84%,结合量为6.9mg/ml介质。这是首次利用小分子亲和配基纯化心脏毒素。与生物大分子配基相比,活性染料分子具有价格便宜,易于合成,性质稳定,不易降解和适合大规模生产等优点。     

The status of tyrosyl residues in a Formosan cobra cardiotoxin.

Spectrophotometric titration of Formosan cobra cardiotoxin showed that two of the three tyrosyl residues were titrated freely with a normal apparent pKa of 9.6 whereas the remaining one ionized at pH above 11.0. Nitration of cardiotoxin in Tris . HCl buffer with tetranitromethane resulted in the selective nitration of tyrosine 11 and tyrosine 22. It also revealed that tyrosine 51 was the abnormal one in the spectrophotometric titration. Complete nitration occurred in the presence of 6.0 M guanidine hydrochloride. Compared with the conformation of native cardiotoxin, the peptide conformation of the partially nitrated cardiotoxin did not change significantly but the conformation of the completely nitrated cardiotoxin changed remarkably. The biological activity of cardiotoxin was indeed affected by nitration, but the immunological activity was nearly intact even when all the tyrosine residues were nitrated.     

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.     

The oxidation of methionine and its effect of the properties of cardiotoxin VII1 from Naja melanoleuca venom.

Methionine residues 24 and 26 of cardiotoxin VII1 from Naja melanoleuca were oxidised to sulphoxides using N-chlorosuccinimide at pH 8.5. The number of equivalents of oxidant required for complete oxidation suggested that the methionine side-chains existed in a relatively "exposed" conformational state in cardiotoxin. The oxidised cardiotoxin was devoid of lethality. It was also non-haemolytic, both on its own and in the presence of phospholipase A2. However, it was still able to precipitate with anti-cardiotoxin antibody. CD studies indicated that the polypeptide backbone conformation was intact in the oxidised cardiotoxin but some perturbation of tyrosine residues was evident. The possibility of a direct or indirect involvement of the methionine residues in the biological activity of the cardiotoxin is discussed.     

Synthesis and inhibitory evaluation of 3-linked imipramines for the exploration of the S2 site of the human serotonin transporter

The human serotonin transporter is the primary target of several antidepressant drugs, and the importance of a primary, high affinity binding site (S1) for antidepressant binding is well documented. The existence of a lower affinity, secondary binding site (S2) has, however, been debated. Herein we report the synthesis of 3-position coupled imipramine ligands from clomipramine using a copper free Sonogashira reaction. Ligand design was inspired by results from docking and steered molecular dynamics simulations, and the ligands were utilized in a structure–activity relationship study of the positional relationship between the S1 and S2 sites. The computer simulations suggested that the S2 site does indeed exist although with lower affinity for imipramine than observed within the S1 site. Additionally, it was possible to dock the 3-linked imipramine analogs into positions which occupy the S1 and the S2 site simultaneously. The structure activity relationship study showed that the shortest ligands were the most potent, and mutations enlarging the proposed S2 site were found to affect the larger ligands positively, while the smaller ligands were mostly unaffected.     

Identification of a α‐helical molten globule intermediate and structural characterization of β‐cardiotoxin,an all β‐sheet protein isolated from the venom of Ophiophagus hannah (king cobra)

β‐Cardiotoxin is a novel member of the snake venom three‐finger toxin (3FTX) family. This is the first exogenous protein to antagonize β‐adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β‐sheet peptides with 60–80 amino acid residues. Here, we describe the three‐dimensional crystal structure of β‐cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β‐cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α‐helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β‐cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.     

Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle

The effects of cardiotoxin fractions from Naja naja kaouthia and Naja naja atra snake venoms and synthetic melittin peptide were examined on lipolytic activity in red blood cells and primary skeletal muscle cultures. Both native cardiotoxin fractions caused considerable production of free fatty acids in red blood cells. This production was abolished when the fractions were first treated with p-bromophenacyl bromide to reduce the venom phospholipase A2 activity contamination. In equine and human primary cultures of skeletal muscle, the N. n. kaouthia cardiotoxin (10 microM) and melittin (2 microM) caused a breakdown of phospholipids and production of free fatty acids and diacylglycerol in the absence of lysophospholipid formation. Additionally, melittin at higher concentrations (10 microM) caused triglyceride breakdown. These studies do not support the suggestion that snake venom cardiotoxins and melittin selectively activate endogenous phospholipase A2 activity. Instead, the toxins primarily activate endogenous phospholipase C activity and, in the case of melittin at high concentrations, triglyceride lipase activity.     

Distribution of Neoparamoeba sp. in sediments around marine finfish farming sites in Tasmania

Marine sediment samples collected from various sites at 2 Atlantic salmon farms in Tasmania were analysed for the presence of Neoparamoeba sp., an amoeba associated with amoebic gill disease (AGD) in farmed Atlantic salmon. Environmental variables of the sediment layer at each site, including redox potential and sulphide concentration, were measured and the general biological condition assessed by video observation. Sediments and environmental data were collected on 4 occasions at each site over a 12 mo period. Neoparamoeba sp. was detected in populations of amoebae recovered by culture from all sites and in 50% of all sediment samples taken. There was evidence of a seasonal influence on the presence of the amoeba, but this was different at each farm. No Neoparamoeba sp. was recovered from any sites at Farm 1 during the winter of 2002 whereas at Farm 2 this was the case for the summer of 2003. There appeared to be no relationship between the presence of Neoparamoeba sp., salmon farming activities and environmental parameters.     

Do cardiotoxins possess a functional site? Structural and chemical modification studies reveal the functional site of the cardiotoxin from Naja nigricollis

Examination of the literature has revealed that regarding the amino acid sequences, cardiotoxins constitute a family of homogeneous compounds. In contrast, cardiotoxins appear heterogeneous as far as their biological and spectroscopic properties are concerned. As a result, comparison between these molecules with a view to establishing structure-activity correlations is complicated. We have therefore reviewed recent works aiming at identifying the functional site of a defined cardiotoxin, ie toxin gamma from the venom of the spitting cobra Naja nigricollis. The biological and structural properties of toxin gamma are first described. In particular, a model depicting the 3-dimensional structure of the toxin studied by NMR spectroscopy is proposed. The toxin polypeptide chain is folded into 3 adjacent loops rich in beta-sheet structure connected to a small globular core containing the 4 disulfide bonds. A number of derivatives chemically modified at a single aromatic or amino group have been prepared. The structure of each derivative was probed by emission fluorescence, circular dichroism and NMR spectroscopy. Also tested was the ability of the derivatives to kill mice, depolarize excitable cell membranes and lyse epithelial cells. Modification of some residues in the first loop, in particular Lys-12 and at the base of the second loop substantially affected biological properties, with no sign of concomitant structural modifications other than local changes. Modifications in other regions much less affected the biological properties of the toxin. A plausible functional site for toxin gamma involving loop I and the base of loop II is presented. It is stressed that the functional site of other cardiotoxins may be different.     

Effects of livestock grazing intensity on soil biota in a semiarid steppe of Inner Mongolia

Intensive livestock is known to significantly affect soil physical and chemical parameters in steppe ecosystems. However, the effects on soil biological parameters still remain unknown. We hypothesized that intensive grazing would significantly decrease the size and diversity of soil biota due to deterioration of the soil environment and reduction in vegetation cover, while the adapted grazing intensity would improve the biological parameters. Soil samples were collected from five sites with different grazing intensities and history in a semiarid steppe of Inner Mongolia in August 2005. Two sites were long-term ungrazed since 1979 (UG79) and 1999 (UG99), one had been moderately grazed in winter (WG), one continuously grazed moderately (CG) and one long-term site was heavily grazed (HG). Soil microbial biomass carbon (C), basal respiration (BR), catabolic diversity of soil microbial communities, protozoa and nematodes abundance were measured. Soil physicochemical variables were also measured to establish the relationships between soil biological parameters and key soil physical and chemical properties. Soil microbial biomass C, BR, biomass specific respiration (qCO₂) and soil protozoa abundance were significantly lower at the HG site compared to the UG79 site, but no clear differences were found in the other sites. However, soil nematodes abundance increased with increasing grazing intensity, and the abundance of soil amoeba were greater in CG than in the other sites. Principal component analysis (PCA) of Biolog data revealed large differences in catabolic capacity of soil microbial communities between UG79, HG and UG99, WG, CG. However, Shannon??s diversity index did not indicate marked effects of grazing intensity on substrate catabolic community structure. In conclusion, heavy grazing negatively affected soil microbial biomass, activity and protozoan abundance, but positively influenced soil nematodes abundance and did not affect soil microbial catabolic diversity. Based on these results, CG may provide an appropriate grazing intensity to be used in the long term in the semiarid steppe of Inner Mongolia.     

The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides

The biological activity of siRNA seems to be influenced by local characteristics of the target RNA, including local RNA folding. Here, we investigated quantitatively the relationship between local target accessibility and the extent of inhibition of the target gene by siRNA. Target accessibility was assessed by a computational approach that had been shown earlier to be consistent with experimental probing of target RNA. Two sites of ICAM-1 mRNA predicted to serve as accessible motifs and one site predicted to adopt an inaccessible structure were chosen to test siRNA constructs for suppression of ICAM-1 gene expression in ECV304 cells. The local target-dependent effectiveness of siRNA was compared with antisense oligonucleotides (asON). The concentration dependency of siRNA-mediated suppression indicates a >1000-fold difference between active siRNAs (IC₅₀ ≈ 0.2–0.5 nM) versus an inactive siRNA (IC₅₀ ≥ 1 µM) which is consistent with the activity pattern of asON when relating target suppression to predicted local target accessibility. The extremely high activity of the siRNA si2B (IC₅₀ = 0.24 nM) indicates that not all siRNAs shown to be active at the usual concentrations of >10–100 nM belong to this highly active species. The observations described here suggest an option to assess target accessibility for siRNA and, thus, support the design of active siRNA constructs. This approach can be automated, work at high throughput and is open to include additional parameters relevant to the biological activity of siRNA.