HPLC Determination of Neurotoxin β-N-oxalyl-L-α, β-diaminopropionic acid and Its α -Isomer in Lathyrus sativus by Precolumn Derivatisation with 1-Fluoro-2,4-dinitrobenzcne

A rapid and simple method is presented for determining β-N-oxalyl-α. β- diaminopropionic acid (β -ODAP) and its much less toxic α -isomer (α -ODAP) in Lathyrus sativus. Seed and foliage extracts of Lathyrus sativus were treated with 1-fluoro-2,4-dinitrobenzene (FDNB) and a reversed-phase high-performance liquid chromatographic method for the separation of the derivatives in the pmol range is reported.

Adaptive Simulated Annealing Based Protein Loop Modeling of Neurotoxins

A loop modeling method, adaptive simulated annealing, for ab initio prediction of protein loop structures, as an optimization problem of searching the global minimum of a given energy function, is proposed. An interface-friendly toolbox—LoopModeller in Windows and Linux systems, VC++ and OpenGL environments is developed for analysis and visualization. Simulation results of three short-chain neurotoxins modeled by LoopModeller show that the method proposed is fast and efficient.

Latest Advances in the Treatment of Post-stroke Limb Spasm with Botulinum Neurotoxin

Objective: To describe the latest progress in the use of botulinum neurotoxin for post-stroke limb spasm. Methods: This paper looks up the relevant research literatures in recent years in PubMed, Web of Science, Springer, Ovid, CNKI, WanFang databases and summarizes them. Results: The latest progress in the use of botulinum neurotoxin for post-stroke limb spasm was studied from the following aspects: the action mechanism of botulinum neurotoxin;efficacy evaluation;injection dose;target muscle selection;guiding technology;combination therapy. Conclusion: Botulinum neurotoxin is the first-line treatment for post-stroke limb spasm. We need to make continuous improvement and progress from the treatment period, injection dose, target muscle selection, guiding technology and efficacy evaluation to improve the quality of life of the majority of post-stroke survivors in China.

Genomic,transcriptomic,and epigenomic analysis of a medicinal snake,Bungarus multicinctus,to provides insights into the origin of Elapidae neurotoxins

The many-banded krait,Bungarus multicinctus,has been recorded as the animal resource of JinQianBaiHuaShe in the Chinese Pharmacopoeia.Characterization of its venoms classified chief phyla of modern animal neurotoxins.However,the evolutionary origin and diversification of its neurotoxins as well as biosynthesis of its active compounds remain largely unknown due to the lack of its high-quality genome.Here,we present the 1.58 Gbp genome of B.multicinctus assembled into 18 chromosomes with contig/scaffold N50 of 7.53 Mbp/149.8 Mbp.Major bungarotoxin-coding genes were clustered within genome by family and found to be associated with ancient local duplications.The truncation of glycosylphosphatidylinositol anchor in the 3'-terminal of a LY6E paralog released modern three-finger toxins(3FTxs)from membrane tethering before the Colubroidea divergence.Subsequent expansion and mutations diversified and recruited these 3FTxs.After the cobra/krait divergence,the modern unit-B ofβ-bungarotoxin emerged with an extra cysteine residue.A subsequent point substitution in unit-A enabled theβ-bungarotoxin covalent linkage.The B.multicinctus gene expression,chromatin topological organization,and histone modification characteristics were featured by transcriptome,proteome,chromatin conformation capture sequencing,and ChIP-seq.The results highlighted that venom production was under a sophisticated regulation.Our findings provide new insights into snake neurotoxin research,meanwhile will facilitate antivenom development,toxin-driven drug discovery and the quality control of JinQianBaiHuaShe.

Single-neuron neurodegeneration as a degenerative model for Parkinson’s disease

The positive effect of levodopa in the treatment of Parkinson’s disease,although it is limited in time and has severe side effects,has encouraged the scientific community to look for new drugs that can stop the neurodegenerative process or even regenerate the neuromelanin-containing dopaminergic nigrostriatal neurons.Successful preclinical studies with coenzyme Q10,mitoquinone,isradipine,nilotinib,TCH346,neurturin,zonisamide,deferiprone,prasinezumab,and cinpanemab prompted clinical trials.However,these failed and after more than 50 years levodopa continues to be the key drug in the treatment of the disease,despite its severe side effects after 4–6 years of chronic treatment.The lack of translated successful results obtained in preclinical investigations based on the use of neurotoxins that do not exist in the human body as new drugs for Parkinson’s disease treatment is a big problem.In our opinion,the cause of these failures lies in the experimental animal models involving neurotoxins that do not exist in the human body,such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine,that induce a very fast,massive and expansive neurodegenerative process,which contrasts with the extremely slow one of neuromelanin-containing dopaminergic neurons.The exceedingly slow progress of the neurodegenerative process of the nigrostriatal neurons in idiopathic Parkinson’s patients is due to(i)a degenerative model in which the neurotoxic effect of an endogenous neurotoxin affects a single neuron,(ii)a neurotoxic event that is not expansive and(iii)the fact that the neurotoxin that triggers the neurodegenerative process is produced inside the neuromelanin-containing dopaminergic neurons.The endogenous neurotoxin that fits this degenerative model involving one single neuron at a time is aminochrome,since it(i)is generated within neuromelanin-containing dopaminergic neurons,(ii)does not cause an expansive neurotoxic effect and(iii)triggers all the mechanisms involved in the neurodegenerative process of the nigrostriatal neurons in idiopathic Parkinson’s disease.In conclusion,based on the hypothesis that the neurodegenerative process of idiopathic Parkinson’s disease corresponds to a single-neuron neurodegeneration model,we must search for molecules that increase the expression of the neuroprotective enzymes DT-diaphorase and glutathione transferase M2-2.It has been observed that the activation of the Kelch-like ECH-associated protein 1/nuclear factor(erythroid-derived 2)-like 2 pathway is associated with the transcriptional activation of the DT-diaphorase and glutathione transferase genes.

A型肉毒毒素注射治疗痉挛性斜颈的引导技术研究进展

痉挛性斜颈(CD)是临床常见的一种局灶性肌张力障碍疾病,主要由于颈部肌肉不自主收缩引起的异常姿势或运动,大多数为特发性。CD的治疗方法有口服药物、A型肉毒毒素(BoNT-A)注射、手术等方法。BoNT-A注射起效快、持续时间长、副作用小、重复性好,是目前首选的治疗方法。然而不同研究中BoNT-A注射的疗效差异较大,为提高BoNT-A注射疗效,临床上各类引导技术应运而生,本文就各类BoNT-A注射引导技术的优势和不足进行综述。

Antidepressant-Like Action of Single Facial Injection of Botulinum Neurotoxin A is Associated with Augmented 5-HT Levels and BDNF/ERK/CREB Pathways in Mouse Brain

The present study was designed to examine the therapeutic effects of Botulinum neurotoxin A(BoNT/A)on depression-like behaviors in mice and to explore the potential mechanisms.These results revealed that a single facial injection of BoNT/A induced a rapid and prolonged improvement of depression-like behaviors in naive and space-restriction-stressed(SRS)mice,reflected by a decreased duration of immobility in behavioral despair tests.BoNT/A significantly increased the 5-hydroxytryptamine(5-HT)levels in several brain regions,including the hippocampus and hypothalamus,in SRS mice.BoNT/A increased the expression of the N-methyl-Daspartate receptor subunits NR1 and NR2 B in the hippocampus,which were significantly decreased in SRS mice.Furthermore,BoNT/A significantly increased the expression of brain-derived neurotrophic factor(BDNF)in the hippocampus,hypothalamus,prefrontal cortex,and amygdala,which were decreased in SRS mice.Finally,BoNT/A transiently increased the levels of phosphorylated extracellular signal-regulated kinase(p-ERK)and cAMPresponse element binding protein(p-CREB),which were suppressed in the hippocampus of SRS mice.Collectively,these results demonstrated that BoNT/A treatment has antidepressant-like activity in mice,and this is associated with increased 5-HT levels and the activation of BDNF/ERK/CREB pathways in the hippocampus,supporting further investigation of BoNT/A therapy in depression.

眼镜蛇神经毒素分离纯化及其经大鼠直肠给药吸收的实验研究

目的利用色谱技术从眼镜蛇粗毒中分离纯化神经毒素,探索神经毒素经大鼠直肠途径给药后的吸收情况和镇痛活性。方法通过CM Sepharose Fast Flow阳离子交换色谱和Sephadex G-50凝胶过滤色谱两步法分离纯化神经毒素。采用免疫组织化学技术观察神经毒素在大鼠直肠组织中的分布。利用Shotgun质谱技术和数据库检测大鼠血浆神经毒素片段。利用HE染色观察神经毒素对大鼠直肠组织结构的影响。利用醋酸扭体法测定神经毒素直肠给药后镇痛活性。结果通过色谱分离纯化得到电泳纯的神经毒素。神经毒素直肠给药3 h后直肠黏膜组织内神经毒素分布明显,阳性面积比率、平均光密度值、H-SCORE值与对照组相比差异明显(P<0.01)。质谱检测到大鼠血浆中神经毒素有关片段。HE染色表明神经毒素对大鼠直肠组织没有损伤,组织结构无明显病理变化。醋酸扭体法测定小鼠直肠给药神经毒素3 h后扭体抑制率达50%,提示具有镇痛活性。结论眼镜蛇神经毒素可以通过直肠吸收入血并且发挥镇痛作用,为后续研究眼镜蛇神经毒素直肠给药制剂提供实验依据。

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.

Exploring the obscure profiles of pharmacological binding sites on voltage-gated sodium channels by BmK neurotoxins

Diverse subtypes of voltage-gated sodium channels(VGSCs)have been found throughout tissues of the brain,muscles and the heart.Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch(BmK)act as sodium channel-specific modulators and have therefore been widely used to study VGSCs.α-type neurotoxins,named BmK I,BmKαIV and BmK abT,bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs.In contrast,β-type neurotoxins,named BmK AS,BmK AS-1,BmK IT and BmK IT2,occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels.Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs,however,indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simpleα-type and β-type neurotoxin distinction.Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region-and/or speciesspecific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs.In this review,we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3-or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.

Discovery of a Potent Botulinum Neurotoxin A Inhibitor ZM299 with Effective Protections in Botulism Mice

Botulinum neurotoxins serotype A(BoNT/A)is the deadliest toxins known to humans and the"Category A"agent for bioterrorism.Over the past 20 years,significant efforts have been put forth to develop effective inhibitors of BoNT/A.Unfortunately,few identified inhibitors possess noteworthy efficacy against BoNT/A in vivo.Here,we performed a high-throughput virtual screening based on the structure-based docking simulations and found a novel potent scaffold 2-thionicotinate that inhibits the BoNT/A light chain(LC).We then synthesized and optimized a novel series of 2-thionicotinate derivatives and comprehensively evaluated their activity against BoNT/A in vitro and in vivo.An optimized compound ZM299 effectively exhibits anti-BoNT/A activity in primary neurons and displayed remarkably therapeutic efficacy against BoNT/A in vivo,which could raise the survival rate of intoxicated mice to 100%(12/12)after lethal doses of BoNT/A exposures.These findings demonstrate that 2-thionicotinates is a promising scaffold for producing more effective anti-BoNT/A analogs,and compound ZM299 is worthy of further preclinical evaluation as a drug candidate for the treatment of botulism.

Construction of an insecticidal baculovirus expressing insect-specific neurotoxin AaIT

Considering the factors which affect gene transcription, translation and the stability of mRNA, without changing the amino acid composition of the encoded polypeptide, AaIT gene encoding insect-specific neurotoxin was designed and synthesized according to bias in codon choice, overall G+C content and G + C content of bases at the third position in codons of polyhedrin genes of baculovirus and of plant genes as well. AaIT gene was fused behind a synthetic gp67 signal sequence and then recombined into the genome of Trichoplusia ni nuclear polyhedrosis virus (TnNPV) by transfer vector pSXIV VI+X3. The recombinant virus TnNPV-AalT (occ+-gal-) was screened. The results of Southern blotting and SDS-PAGE demonstrated that AaIT gene had integrated into the genome of virus and expressed. Bioassays on the 3rd-instar Trichoplusia ni larvae showed that recombinant viruses TnNPV-AalT could shorten the time of killing insect and improve the efficiency of killing agronomically important insects.

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

Preliminary crystallographic studies of a neurotoxin with a high toxicity from Chinese scorpion Buthus martensii Karsch

Scorpion toxins are a family of small neurotoxic proteins with high selectivity. Gener-ally, most of the toxins are a group of basic homologous polypeptides containing about60—80 amino acids except BmK Ⅳ whose pl is 5.3. It has been reported that theybind to various ion channels with high affinity and selectivity. In spite of the significantsequence similarity found among different toxins, they display various degrees of toxicityand specificity to different animal species. So far they form a good system to study thestructure-function relationship.

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.

α-神经毒素的分离纯化及其经小鼠直肠给药后肠道体外荧光成像分析

目的从眼镜蛇粗毒中分离纯化短链α-神经毒素活性成分,观察荧光染料标记的α-神经毒素经小鼠直肠给药后的肠道内分布和动态过程。方法使用CM Sepharose Fast Flow阳离子交换层析和Sephadex G-50凝胶过滤层析分离纯化电泳纯的α-神经毒素。将4只C57小鼠分为对照组和实验组,对照组采用荧光染料CY7-SE标记,实验组采用荧光染料CY7-SE与α-神经毒素相互结合。使用成像仪检测CY7-SE标记的α-神经毒素在直肠给药1、3 h后肠道内的荧光强度分布情况。结果分离纯化得到电泳纯的α-神经毒素。α-神经毒素直肠给药1 h后,实验组相较于对照组的小肠段和胃的荧光强度较强,盲肠、结肠段荧光强度较弱。α-神经毒素直肠给药3 h后,实验组相较于对照组的小肠、盲肠、结肠段和胃的荧光强度均较弱。结论从眼镜蛇粗毒中分离纯化得到的α-神经毒素经直肠给药可先富集于小鼠小肠段及胃,3 h后被肠道黏膜吸收代谢。本研究为开发神经毒素的直肠递送制剂提供了实验依据。

血清嗜酸性粒细胞衍生神经毒素与儿童哮喘急性加重风险和严重程度的关系

目的探讨血清嗜酸性粒细胞衍生神经毒素(EDN)与儿童哮喘急性加重风险和严重程度的关系。方法选取2018年6月—2020年6月期间衡水市妇幼保健院儿科收治的117例哮喘儿童,包括52例哮喘缓解期儿童和65例哮喘急性加重期儿童。另外,同期接受健康检查的70例健康儿童被纳入健康对照组。按哮喘急性加重严重程度将哮喘急性加重患儿分为轻度组、中度组和重度组。采用酶联免疫吸附法检测血清EDN水平。采用多因素Logistic回归和受试者工作特征(ROC)曲线分析血清EDN与哮喘急性加重的关系。结果与健康对照组相比,哮喘缓解组和急性加重组患儿血清EDN水平均升高,且急性加重组较缓解组升高更显著(P<0.05)。在哮喘急性加重儿童中,中度组和重度组患儿血清EDN水平较轻度组均升高,且重度组较轻度组升高更显著(P<0.05)。血清EDN(OR=1.172,95%CI=1.042~1.319,P=0.008)、EOS(OR=60.238,95%CI=1.223~2966.959,P=0.039)、IgE(OR=1.063,95%CI=1.014~1.115,P=0.011)、FEV_(1)/FVC(OR=0.881,95%CI=0.776~1.000,P=0.049)是哮喘急性加重的独立影响因素(P<0.05)。血清EDN水平区分重度和轻中度哮喘急性加重儿童的AUC为0.880(95%CI:0.778~0.982)。经Spearman法分析,哮喘急性加重儿童血清EDN和白细胞计数(WBC)、嗜酸性粒细胞(EOS)计数、EOS%、免疫球蛋白E(IgE)、C反应蛋白(CRP)呈正相关(P<0.05),与患儿1秒用力呼气容积占预计值的百分比(FEV_(1)%pred)、用力肺活量占预计值的百分比(FVC%pred)、FEV_(1)/FVC均呈负相关(P<0.05)。结论血清EDN上调与儿童哮喘急性加重风险和病情严重程度增加有关。血清EDN可能是儿童哮喘急性加重风险和病情严重程度监测的良好生物标志物。

Crystallization adn molecular aggregation of presynaptic neurotoxin from Agkistrodon halys pallas

Phospholipase A2 catalyzes specially the hydrolysis of the ester bond at the C2 position of3-sn-phosphoglycerides. Besides the enzymatic activity, the venom phospholipase A2 from varioussources displays complicated pharmacological activity and toxicity. The phospholipaseA2 neurotoxin is a noteworthy group in all phospholipase A2 species. It has been

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