我国不同地区东亚钳蝎(Buthus martensii Karsch)毒在三种类型电泳中的比较研究

用SDS—不连续聚丙烯酰胺凝胶板电泳、pH4.5—不连续聚丙烯酰胺凝胶板电泳和聚丙烯酰胺凝胶圆盘等电聚焦电泳,分离我国不同地区东亚钳蝎毒。其图谱表明:不同产地的东亚钳蝎毒蛋白的种类及含量存在一定的差异,地区相近,差异较小,反之亦然。

Effect of scorpion venom analgesic active peptide extracted from Buthus martensii Karsch on evoked potential in the thalamic posterior nucleus group in rats

BACKGROUND： Buthus martensii Karsch is a rare medicinal animal, and dried integral Buthus rnartensii Karsch is an important drug in traditional Chinese medicine. OBJECTIVE： To investigate the effects of scorpion venom analgesic active peptide （SAP） extracted from Buthus martensii Karsch on evoked unit discharge of the common peroneal nerve in the posterior nucleus group of the thalamus using a stereotaxic electrophysiological extracellular microelectrode recording. DESIGN, TIME AND SETTING： One-way designed study, performed in the Physiological Laboratory of Shenyang Medical College on September 15, 2006. MATERIALS： Fifty 3-4 months old Wistar rats （25 males and 25 females） were used. SAP was provided by Shenyang Pharmaceutical University. Morphine solution was made by the First Drug Manufactory, Northeastern Drug Manufacture Group （batch number： H20013351）. Naloxone solution was made by Hunan Pharmaceutical Co., Ltd. （batch number： H43021669）. Type ATAC-350 medical data processing equipment was made by the Photoelectricity Company, Japan. METHODS： Fifty rats were randomly divided into the SAP group （n=20）, saline group （n=10）, morphine group （n=10）, or naloxone group （n=10）. In the SAP group, the common peroneal nerve was separated and stimulated with a single square wave （17-19 V intensity; 0.2 ms width; 20 ms retardation time）. Subsequently, SAP （0.01%, 2 μL） was injected into the posterior nucleus group of the thalamus. Rats in the naloxone group were injected with naloxone （1.0 mg/kg i.v.） before SAP injection. Rats in the saline group and the morphine group were injected with saline （2 μL） or morphine （0.01%, 2μL）, respectively, before SAP injection. Other procedures were the same as those in the SAP group. MAIN OUTCOME MEASURES： Evoked discharge in the posterior nucleus group of the thalamus and effects of SAP alone and SAP in combination with saline, morphine, or naloxone on discharges in the posterior nucleus group of the thalamus as measured by TQ-19 medical data processing equipment. RESULTS： SAP group： At 1-3 minutes after SAP injection, evoked discharges in the posterior nucleus group of the thalamus were inhibited, and the inhibitory time lasted for （45.0±0.7） minutes. Saline group： Evoked discharges in the posterior nucleus group of the thalamus did not change after saline injection. Morphine group： At 1-3 minutes after morphine injection, evoked discharges in the posterior nucleus group of the thalamus were inhibited, and the inhibitory time lasted for （35.0±7.8） minutes. Naloxone group： SAP had no effects on evoked potentials in the posterior nucleus group of the thalamus. CONCLUSION： The inhibitory effect of SAP on evoked potentials was superior to that of morphine at the same concentration （2 μL of 0.01% solution）. Naloxone resupination demonstrated that the inhibitory effects of SAP on evoked discharges were influenced by the opioid receptor.

The effects of scorpion(Buthus martensi Karsch)venom on rat left ventricular function

In order to investigate the cardiovascular effects of the scorpion(Buthus martensiKarsch)venom(BmKv),the left ventricle of the rats was catheterized via the right carotidartery.The LVP,LVEDP,+dp/dt max,Vmax,HR and BP were observed.The results showedthat intravenous injection of the BmKv(60μg/kg),in comparison with the control,elicited obvi-ous hypertension and increase of cardiac contractility,both of which lasted for 1h,while theheart rate had no significant change rand that pretreating the rats with alpha-adrenergic blocker,phentolamine,antagonized the hypertensive effects,but did not antagonize the increase of cardiaccontractility.Pretreatment with beta-adrenergic blocker,propranolol,has no influence on the ef-fects of the venom.It is suggested that the hypertensive effects are due to the activation of al-pha-adrenergic receptor,whereas the increase of cardiac contractility may not be resulted fromthe activation of beta-adrenergic receptor.The BmKv treated with dithiothreitol before injectionhad no cardiovascular effects,indicating that the intact disulfide bridges play a decisive role inthe cardiovascular effects of the BmKv.

Amino Acid Sequence of an Excitatory Insect-selective Toxin (BmK IT) From Venom of the Scorpion Buthus martensi Karsch

The insect-selective neurotoxin(BmK IT) of scorpion Buthus martensi Karsch was first reduced and S-alkylated, and then digested by TPCK-trypsin and Staphylococcus aureus V-8 Protease. The enzymatic peptides were purified on TLC-plastic sheet and submitted to determine their amino acid compositions and sequences. The sequence of the 70 amino acid residues of BmK IT was established with reference to the primary structure of AaH IT, another excitatory insect-selective toxin from the venom of North African scorpion Androctonus australis Hector. About 75% of the homologous sequence was found in the molecules of BmK IT and AaH IT. It is obvious that the results contribute toward better understanding of the molecular structure characteristics, structure/activity relationship of scorpion insect-selective toxins, and they can serve as the molecular basis for utilizing the toxins as a tool to clarify molecular mechanism involved in channel gating, and to infer the possibility of developing them as new selective bioinsecticides.

Cloning and sequencing of two depressant insect selective neurotoxin cDNAs from Buthus martensii Karsch

During evolution, scorpions have developed the ability to produce a series of toxins. Scorpion toxins, which are reserved in terminal segments of scorpion, serve as the arms for predation and self-defence. They have also been used as medicine for some human sickness. As far as the targets that they act on are concerned, these toxins can be

Molecular Characteristics of Four New Depressant Insect Neurotoxins Purified From Venom of Buthus martensi Karsch by HPLC

Suitable pattern and high yield were obtained when the reverse-phase performance liquidchromatography (RP-HPLC) was used to separate neurotoxins from venom of Chinese scorpion Buthusmartensi Karsch.Using this technique,the venom was first separated to two main regions.The toxicitytests show that the insect-selective neurotoxical components are concentrated in the latter region,from whichfive insect-selective neurotoxins designated by BmK IT1-IT5 were obtained.According to the results of thetoxicity test as well as the amino acid composition and N-terminal analyses,BmK IT1 is the excitatory insectneurotoxin as reported in a previous paper,and the others are the newly found depressant insect-selectiveneurotoxins.The molecules of all the four toxins are single-chain minipeptides of about 60 amino acids.Their isoelectric points (pI) are between 8.3 and 8.5.The fact that BmK IT2 loses completely its insect tox-icity after being modified by fluorochrome shows that the positive charges on the molecular surface of thiskind of toxins are important to maintaining the bioactivity of the molecules.

抗痛蝎多肽的分离与筛选

东亚钳蝎(Buthusmartensii Karsch)是我国重要的中药材之一,其毒液富含多种离子通道及受体的调制分子,对疼痛,癫痫等疾病有治疗作用。因此,针对蝎毒素中复杂多肽的分离、筛选与鉴定是蝎毒素研究与开发的重点。为了分离并筛选出具有镇痛效果的蝎毒多肽,实验利用反相高效液相色谱的方法分离纯化蝎毒,将得到的4种蝎毒多肽组分分别命名为BmKⅡ-10、BmKⅡ-11、BmKⅡ-16和BmKⅡ-24,结合质谱技术鉴定其分子量分别为7530.8 Da、7366.0 Da、5949.6 Da和6852.4 Da。通过构建疼痛模型检测蝎毒多肽组分对小鼠疼痛的影响,结果显示,BmKⅡ-10,BmKⅡ-11可能为蝎毒中的致痛组分,而BmKⅡ-16具有显著的镇痛效果,能有效抑制由福尔马林和角叉菜胶引起的自发痛与机械痛行为。本研究所采用的多肽分离纯化方法能有效获得蝎毒素中多种多肽组分,进一步验证发现部分组分与疼痛密切相关,为疼痛药物的开发与应用提供了理论和实验依据。

Primary Structure of Depressant Insect-selective Neurotoxin From Venom of Scorpion Buthus martensi Karsch

Scorpion anti-insect toxins can be divided into long chain (about 61-70 aminoacid residues)and short chain (about 5 amino acid residues) types according to theirmolecular size, and the former can be further divided into excitatory and depressanttypes on the basis of their pharmacological action. In our previous papers, the iso-lation and determination of the primary structure of an excitatory

Crystal structure determination of an acidic neurotoxin (BmK M8) from scorpion Buthm martensii Karsch at 0.25nm resolution

The crystal structure of an acidic neurotoxin, BmK M8, from Chinese scorpion Buthus martensii Karsch was determined at 0.25 nm resolution. The X-ray diffraction data of BmK M8 crystals at 0.25nm resolution were collected on a Siemens area detector. Using molecular replacement method with a basic scorpion toxin AaH II in a search model, the cross-rotation function, PC-refinement and translation function were calculated by X-PLOR program package. The correct orientation and position of BmK M8 molecule in crystal were determined in a resolution range of 1.5 - 0.35nm, The oystallographic refinement was further performed by stereo-chemical restrict least-square technique, followed by simulated annealing, slow-cooling protocols. The final crystallographic R-factor at 0.8-0.25 nm is 0.171. The standard deviations of bond length and bond angle from ideality are 0.001 7nm and 2.24° , respectively. The final model of BmK M8 structure is composed of a dense core of secondary structure elements by a stretch of α-helix with two and a half turns (residues 19-28) and a three-stranded antiparallel β-sheet (residues 2-4, 32 - 37, 45- 51). In addition, three loops protruded from the structural core. The general folding properties of BmK M8 molecule were described; a common structure motif which may appear in all scorpion neurotoxins was identified. The conserved aromatic residues and charged residues were found to be distributed on two roughly opposite surfaces of the molecule. The relationship between these two faces and receptor-binding sites are also discussed.

Molecular characterization of a K+ channel blocker in the scorpion Buthus martensii Karsch

K+ channel blockers of scorpion venoms are of important value in studyingpharmacology and physiology of specific K+ channel of celis. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca2+-activated K+ channel blocker, two 'back to back' degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpion Buthus martensii. The cDNA is composed of 3 parts: 5’ UTR, oRF and 3’ UTR. The flanking sequence of translation initiation codon ATG is AAAKTGA, which is highly conserved in scorpion Na+ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3’ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is

Two non-coding transcripts of toxins from the scorpion But-hus martensii Karsch: Evidence for the existence of NMD in scorpion venom gland cells

Two non-coding transcripts of toxins were iso-lated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch, named BmαTX15-SP and BmTXKβ-SP, respectively. Analysis of nucleotide sequences shows that their 5’ sequences are identical to the 5 ’UTR and upstream open reading frame (ORF) sequences of the cDNAs encoding BmαTX15 and BmTXKb, two toxins from Buthus martensii Karsch. But no detectable homology exists in other regions. Two polyadenylated non-coding transcripts encode multiple short ORFs with no more than 34 amino acid resi-dues. The tailing signal (AATAAA) is situated at 14 or 18 bp downstream from the poly(A). The data of genomic se-quences provides support for Bmα TX15-SP and BmTXK β-SP not deriving from splicing errors of pre- mRNAs. Our finding provides evidence for the existence of non-sense-mediated mRNA decay (NMD) pathway in scorpion venom gland cells.

HPLC Purification and Partial Amino Acid Sequence of an α-type Insect Neurotoxin From Venom of Scorpion Buthus martensi Karsch

Scorpion venoms contain several kinds of neurotoxins, such as antimammalian neurotoxins, anti-insect neurotoxins and others. But most of them form a family of structurally related single chain proteins of 60—70 amino acid residues and selectively interact with voltage-dependent sodium channels in different excitable cells, only a few minipeptides of 31—39 amino acid residues are proved to block potassium channels. As a kind of molecular probe, scorpion neurotoxins have been widely used for analyzing the

Purification,Characterization and ~1H NMR Resonance Assignment of an α-Like Neurotoxin BmK 16 from the Venom of Chinese Scorpion Buthus martensii Karsch

A natural scorpion toxin BmK 16 was purified for the first time from the venom of the Chinese scorpion Buthus martensii Karsch (BmK) by using combined gel filtration, ion exchange and reversed phase chromatography. The sequence of the N terminal 8 amino acid residues was determined by Edman degradation. Using the N terminal sequence as a tag, the database searching revealed a hit in the scorpion cDNA Bank. The sequence for N terminal 8 amino acid residues, molecular weight and amino acid compositions of BmK 16 were identical with the calculated values according to the first 64 residues' sequence of the precursor peptide alpha neurotoxin TX16 derived from the sequence of the cDNA AF156597 (EMBL). The sequence specific resonance assignment of BmK 16 was achieved and the intact sequence of BmK 16 was determined as followings: VRDAY IAKPH NCVYE CARNE YCNDL CTKNG AKSGY CQWVG KYGNG CWCKE LPDNV PIRVP GKCH. Furthermore, the results from the sequence homology analysis and the toxicity assays indicated that BmK 16 was an α like scorpion neurotoxin.

药用全蝎毒液的蛋白差异分析

目的:药用全蝎市场需求旺盛,导致市面上出现了大量易混肴种,急需对基原性状与成分差异进行评价。方法:通过收集不同产区资源,鉴定基原与蛋白成分差异,评价其性状优劣。结果:经鉴定,采集的蝎子除东亚钳蝎外,还鉴定出细尖狼蝎、条纹木蝎;就毒囊来看,东亚钳蝎的毒囊尺寸与蛋白含量最优,为422.87μm×293.74μm与(601.6±432.3)μg/mL;而对抗癫痫肽类成分进行分析,东亚钳蝎中Na^(+)/K^(+)毒素肽、蝎毒素类物质含量最高,其次为细尖狼蝎,而条纹木蝎中仅对部分Ca^(2+)毒素肽存在较高表达。结论:东亚钳蝎在毒囊尺寸与蛋白成分上均要优于其他蝎种,是全蝎类药材的优质基原。

东亚钳蝎及蝎毒镇痛作用研究进展

目的综述东亚钳蝎及蝎毒的镇痛作用、蝎毒镇痛活性肽及其突变体的镇痛活性,为进一步研究蝎毒镇痛作用提供参考。方法参考国内外74篇文献,从全蝎和蝎毒的镇痛作用、蝎毒的镇痛成分、蝎镇痛活性肽及其突变体三个方面进行总结和综述。结果全蝎和蝎毒均具有较好镇痛效果,但临床直接应用会产生很大的毒副作用。为减少这种副作用,现已从蝎毒中分离纯化出11类单一的具有镇痛作用的活性成分。此外,国内许多研究者用蝎毒分离纯化出的9种蝎镇痛活性肽作为先导化合物进行修饰改造,并探究其突变体的镇痛活性以及影响镇痛效果的结构,为研发出新型镇痛药物提供了理论依据。结论蝎毒及其活性成分不仅对内脏痛、躯体痛、癌肿痛、神经痛等有较好的疗效,对多种急、慢性疼痛均有较强抑制作用,有助修复受损神经,而且镇痛效果强于吗啡,又无成瘾性,为开发出具有无成瘾性、持久性、安全性较高的新型镇痛药物提供了理论依据。

^(60)Co-γ射线辐照灭菌对全蝎药粉品质的影响研究

目的研究不同剂量^(60)Co-γ射线辐照灭菌对全蝎药粉成分及卫生学指标的影响,为全蝎灭菌工艺提升提供依据。方法使用4、6、8、10 kGy^(60)Co-γ射线对全蝎药粉进行辐照灭菌,对灭菌前后样品的水分、总灰分、酸不溶性灰分、黄曲霉毒素、酸溶性浸出物、重金属及有害元素等药典要求检查项进行测定,以及对微生物含量等卫生学指标进行测定,并对不同剂量^(60)Co-γ射线辐照灭菌的全蝎药粉进行HPLC成分含量分析。结果不同剂量^(60)Co-γ射线辐照前后全蝎药粉中的水分、总灰分、酸不溶性灰分、黄曲霉毒素、酸溶性浸出物、重金属及有害元素无明显差异;全蝎药粉在不同剂量^(60)Co-γ射线辐照灭菌前后,各试验组之间卫生学指标差异极显著,4 kGy及以上的^(60)Co-γ射线动态辐照可对微生物进行有效杀灭,6 kGy及以上的剂量灭菌效果好;高效液相色谱测定灭菌前后5种核苷类成分及17种氨基酸含量无显著差异,灭菌前后质量稳定。结论^(60)Co-γ射线辐照可有效杀灭全蝎药粉中微生物,6~10 kGy的剂量效果最佳,辐照灭菌前后药粉药典检查项无明显差异,辐照后的药粉品质更优。

一种来源于蜘蛛毒液的新型Nav1.7多肽抑制剂的发现与鉴定

Nav1.7特异性地表达在外周伤害性感觉神经元。临床遗传学、动物模型和药理学研究表明,Nav1.7是一个重要的镇痛药物开发靶点。本研究以沟纹硬皮地蛛(Calommata signata Karsch)毒液为研究对象,通过电刺激采集毒液、反相高效液相色谱分离纯化、质谱鉴定、蛋白质测序以及全细胞膜片钳记录等方法,筛选并鉴定到一种对Nav1.7有抑制作用的新型多肽毒素,命名为APTX-1。质谱鉴定该多肽毒素的分子质量为7815.2 Da;其N端前10位氨基酸序列为ASCKQVGEEC。APTX-1抑制Nav1.7电流具有浓度依赖性,其半数抑制浓度为(0.46±0.08)μmol/L。通道动力学分析显示,APTX-1并不影响Nav1.7的翻转电位、电压依赖性稳态激活曲线和失活曲线,表明该多肽毒素并不影响Nav1.7的离子选择性和电压依赖性。综上所述,本研究获得了一个新型Nav1.7调制剂,并探究了其对Nav1.7的作用特点,为靶向Nav1.7的镇痛药物研发提供了潜在先导分子。

东亚钳蝎昆虫毒素BmKIT的cDNA序列分析

利用ＲＴ－ＰＣＲ方法从东亚钳蝎（ＢｕｔｈｕｓｍａｒｔｅｎｓｉＫａｒｓｃｈ，ＢｍＫ）的尾腺总ＲＮＡ中扩增出挛缩型昆虫毒素ＢｍＫＩＴ的ｃＤＮＡ，并对其核苷酸序列进行了分析。将含该基因的重组分泌型表达质粒ｐＥｘＳｅｃＩ－ＢｍＫＩＴ转化大肠杆菌ＢＬ２１（ＤＥ３），表达出分泌型的融合蛋白。

全蝎化学成分和药理作用的研究进展

全蝎为蛛形纲蝎目钳蝎科昆虫,为息风平肝的要药,味辛、平,有毒。其药用历史悠久,具息风镇痉、通络止痛、攻毒散结之功效,主要用于治疗风湿、癫痫、中风、慢性疼痛等疾病。其主要活性成分为蝎毒、甾体衍生物、生物碱等。全蝎具镇痛抗炎、抗癌、抗癫痫、抗惊厥、抗凝、抗菌、促生长、提高免疫力等药理作用。本研究对全蝎化学成分、药理作用进行综述,以期为全蝎的深入研究和开发利用提供参考。

全蝎蛋白抗肿瘤活性研究

目的比较全蝎蛋白超滤分段后各分子量段对肺腺癌细胞A549的抑制作用,筛选有效部分。方法采用蛋白酶酶解法提取全蝎总蛋白,利用80%乙醇沉淀并利用改良Lowry法测定蛋白质含量,再经超滤进行分段、冷冻干燥后通过MTT法研究其对肺腺癌细胞(A549)的抑制作用。结果 80%乙醇沉淀全蝎蛋白具有较高的选择性,其<3 kDa分子量段对A549细胞无抑制作用,3~10 kDa分子量段对A549细胞的抑制作用不明显,>10 kDa分子量段对A549细胞具有显著的抑制作用,IC_(50)=204.7μg·mL^(-1)。结论 80%乙醇沉淀物中蛋白质含量为89.4%;>10 kDa分子量段全蝎蛋白对A549细胞具有显著的抑制作用。