The peptide fraction of Bothrops jararaca snake venom induces toxicological effects on the male reproductive system after local envenomation in mice

Bothrops envenomation is complex and provokes prominent local tissue damage and systemic disturbances,but little is known about their effects on the male reproductive system.After intratesticular injection,the bioactive peptide fraction(Bj-PF)obtained from Bothrops jararaca snake venom changes the structure of different stages of the seminiferous epithelium cycle in adult mice.For the first time,we investigated whether local envenomation of Bj-PF induces toxicological effects on the male reproductive system,particularly on the seminiferous epithelium and Sertoli cells.Male adult mice were treated with 0.24 mg.kg^(-1) by intramuscular(i.m.)injection for 24 h.The testes samples were collected for morphological and morphometric evaluation.The toxicological effects of Bj-PF were also analyzed on mitochondrial metabolism and nitrite(NO2)production in 15P-1 Sertoli cell culture.Bj-PF changed the structure and function of the seminiferous epithelium,particularly the disruption of the epithelium and the presence of degenerated germ cells in the adluminal compartment,but there were no alterations in the basal compartment.Bj-PF increased the thickness of the seminiferous epithelium and decreased the lumen diameter of the tubule.Semiquantitative histological assessment of the degree of tubule degeneration revealed that Bj-PF also increased the number of hypospermatogenic tubules compared to control.Bj-PF reduced NO2 levels in 15P-1 Sertoli cells without changing the mitochondrial metabolism.Overall,the fact that Bj-PF alters the structure and function of the seminiferous epithelium suggests that bioactive peptides found in B.jararaca snake venom can have toxicological effects on the reproductive systems of affected male mice,providing new insight into the biological characteristics of snake venom and therapeutic strategies for envenomation inflammation.

Therapeutic potential of snake venom in cancer therapy:current perspectives

Many active secretions produced by animals have been employed in the development of new drugs to treat diseases such as hypertension and cancer.Snake venom toxins coutributed significantly to the treatment of many medical conditions.There are many published studies describing and elucidating the anti-cancer potential of snake venom.Cancer therapy is one of the main areas for the use of protein peptides and enzymes originating from animals of different species.Some of these proteins or peptides and enzymes from snake venom when isolated and evaluated may bind specifically to cancer cell membranes,affecting the migration and proliferation of these cells.Some of substances found in the snake venom present a great potential as anti-tumor agent.In this review,we presented the main results of recent years of research involving the active compounds of snake venom that have anticancer activity.

Applications of snake venoms in treatment of cancer

Snake venoms are folk medicines used since ages. The components of snake venoms have high specific affinity and actions on cells and cell components. Also snake venoms are largely cytotoxic to tumor cells than normal cells. In addition to these, they have several therapeutic actions that make them an attractive option in the management of cancer. The advent of modern technologies has greatly helped in extracting and identifying new components of therapeutic interests in short time. The article highlights the importance of snake venoms in the management of cancer, so as to motivate curious researchers to devote their skills in this fascinating area. This in turn may bring hope, smile and relief to several cancer patients in future.

PURIFICATION AND CHARACTERIZATION OF A NEW FIBRINOGENASE FROM THE VENOM OF THE CHINESE HABU SNAKE (Trimeresurus mucrosquamatus)

A new fibrinogenase(EC 3.4.2 I.5)was isolated and purified from the venom of Chinese habu snake(Trimeresurus研ucrosg“ama似s)by DEAE—SephadexA-50,DEAE—Sepharose CL一6B,MonoQ(FPLC)CG.1umn chromatography.It showed a single protein band both in sodium dodeeyl sulfate(SDS)·polyacrylami-de geI electrophoresis and alkaline polyacrylamide gel electrophoresis.The molecular weight was estimated tobe 26000 by SDS·p01yacrylam|de gel electrophoresis.The isoeleetric point was found to be pH 4.7.Itwas a glycoprotein containing 6.4‘%carbohydrate with o.3%neutral sugar,1.2%sialic acid,4.9%he.xosamine.It was composed of about 1 78 amino acid residues and rich in glycine and aspartic acid.Thefibrinogenase of the venom of T.munro$quclmatu$TWV№was heat stable but labile to acid.Its extinctioncoefficient(1mg/m1)at 280rim was 1.558.Purified TMVFg had strong arginine esterase activity·the Kmto benzoylarginine ethylester(BAEE)was 1.4×1 0一M.The enzyme activity could be inhibited by pheny.1mefha口esulfonyfluoride(PMSF),but was not affected by ethylenediamine tetraacetlc acid(EDTA).TMVFghad fibrinogenolytie activityl electrophoresis of fibrinogen degraded with TMVFg revealed the rapiddisappearance of the口(alpba)and B(beta)‘chains and the appearance I】f lower molecular weight frag.merits.TMVFg did not cause fibrinogen solution clotting,nor coagulating plasma and showed n^ither hemorr-hagic activity nor proteolytic activity toward casein.TMVFg had activati^lg fibrinolytic activity

Global Insight into N-Glycome and N-Glycoproteome of Three Most Abundant Snake Venoms in Asia

Snake venom is a complex cocktail including a variety of biological active proteins and proteinaceous components, which have considerable medical and pharmacological importance. N-Glycosylation is widely impli- cated as a common modification in numerous venom proteins and impacts the in vivo venomic functions. However, systematic survey of N-glycome and N-glycoproteome on snake venoms has not been undertaken. In this study, em- ploying combination of N-glycomics and N-glycoproteomics strategies, we explored the N-glycosylation including both N-glycoproteins and N-glyco-chains in three venoms from Agkistrodon blomhoffii, Naja naja atra Cantor and Vipera russelii siamensis Smith, respectively, which are amongst the most abundant venomous snakes in Asia. As a result, numbers of N-glycoproteins and N-glycans were identified. However, the overlaps of N-glycoproteins and N-glycans of the three venoms were small. Thus, the exploration results of N-glycome and N-glycoproteome indicate that N-glycosylation increases the complexity and variety of the three venoms. Our research provided some new horizons for the comprehensive understanding of venoms variation, which is helpful for the basic venom re- search as well as the management of snake envenomation.

0.27-nm resolution crystal structure of Haemorrhagin I from snake venom of Agkistrodon acutus

THE snake venom zinc-metalloproteinases and matrix metalloproteinases (MMP) have the similar active site and biological function. They all belong to the MMP super-family. Some

Crystal structure determination of alkaline haemorrhagin AaHⅢ from snake venom of Agkistrodon acutus

The haemorrhagin AaH Ⅲ isolated from the snake venom of Agkistrodon acutus is one of the few al-kaline ones in snake venoms. Its crystals belong to space group P212121 with a = 9. 573 4 nm, b = 4. 996 7 nm and c = 4. 728 8 nm. Its crystal structure was determined by the molecular replacement method according to the model of metalloproteinase Adamalysin n from eastern rattlesnake venom. The AaHⅢ structure has been refined by PROLSQ. The final R factor was 0.254 and the RMS deviations of bond lengths and angles were 0. 001 8 nm and 1.5°. The structure comparison suggested that AaHⅢ has a similar structure to other snake venom zinc-metalloproteinases. They all belong to matrix metalloproteinases super-family.

Expression, refolding and preliminary characterization of recombinant snake venom metalloproteinases: Implica- tion for the hemorrhagic mechanism

Two cDNAs encoding hemorrhagic snake venom metalloproteinase acutolysin A and non-hemorrhagic metalloproteinase (BR) were cloned into the expression vector pET-22b, respectively, and the corresponding two recombinant proteins, A-22b and BR-22b, were produced in inclusion bodies in E. coli BL21(DE3). The recombinant proteins were then subjected to solubilization, purification and refolding in vitro. A-22b showed hemorrhagic activity but no detectable proteolytic activities toward fibrinogen and fibronectin. Natural acutolysin A had both hemorrhagic activity and proteolytic activity toward these substrates. BR-22b showed the proteolytic activities toward fibrinogen, but no hemorrhagic activity. In addition, two chimeric genes, C1 and C2, were constructed and cloned into pET-22b, and the corresponding recombinant proteins, C1-22b and C2-22b, were also expressed in inclusion bodies. C1-22b involved N-terminal 110 amino acids of BR and C-terminal 95 amino acids of acutolysin A, while C2-22b contained N-terminal 108 amino acids of acutolysin A and C-terminal 112 amino acids of BR. The biological activities of C2-22b and C1-22b were similar to those of A-22b and BR-22b, respectively. Our results suggested that N-terminal major subdomain of a snake venom metalloproteinase might play a key role in hemorrhagic activity and have an appreciable effect on the selectivity for protein substrates.

Matrix Assisted Laser Desorption lonization Mass Spectrometric Method for the Separation and Molecular Weight Determination of Snake Venom Proteins

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/MS) is a new, powerful analytical tool for the investigation of large biomolecules. Since its inception in 1986 by Koichi Tanaka and Franz Hillenkamp separately, MALDI/MS has been successfully applied to the investigation of

Preliminary X-ray diffraction study on haemorrhagin AaHⅢ from snake venom of Agkistrodon acutus

Haemorrhagins,Which cause local haemorrhage or even death after injection into exper-imental animals,exist widely in many kinds of snake venoms.Almost allthe haemorrhagins reported are zinc-metalloproteinases with highly conserved amino acid se-quences.Some of them could degrade the proteins in matrices so that they are the im-portant targets for drugs to combat diseases such as arthritis and cancer.Snake venommetalloproteinases can be divided into three classes based on their molecular

Purification and Characterization of Cytotoxins from Agkistrodon acutus Venom and Their Anticancer Activity

Aim To investigate the anticancer activity of two new cytotoxins from thevenom of Agkistrodon acutus. Methods The venom was isolated by FPLC column chromatography consistingof DEAE Sepharose FF and Source 30S. The cytotoxic activity on tumor cells was detected by MITmethod. Purity and molecular weight were determined by SDS-PAGE (silver staining). Their stabilitiesto temperature and pH were also detected. Results Two pure cytotoxins named ACTX-6 and ACTX-8 wereobtained. Their molecular weights are 98 kDa and 27 kDa, respectively. ACTX-6 consists of twosubunits bonded together by disulfide bonds. Conclusion ACTX-6 and ATCX-8 have highest inhibitoryactivity on lung cancer cell A549. ACTX-6 is stable to heat while ACTX-8 not. ACTX-6 is stablebetween pH 7-9 and ACTX-8 between pH 6 - 9.

Changes in hippocampal histamine receptors in rats after treatment with Trimeresurus albolabris venom

BACKGROUND： It has been demonstrated that histamine and its receptors in the hippocampus play an important role in memory and/or learning behaviors.OBJECTIVE： To investigate the expression levels of the histamine receptor gene and protein in the hippocampi of rats prior to and after administration of Trimeresurus albolabris venom using reverse transcription-polymerase chain reaction （RT-PCR） and Western blot techniques. DESIGN, TIME AND SETTING： A controlled observation based on cellular protein level was performed in the College of Life Sciences, Chongqing Normal University between March 2005 and April 2007. MATERIALS： Eighty adult male Sprague-Dawley rats were provided by the Laboratory Animal Center of the Third Military Medical University of Chinese PLA. The lyophilized powder of Trimeresurus albolabris venom was collected from Jin-Hu-Shan in Chongqing, China. METHODS： Twenty rats were randomly and evenly divided into an experimental group and a control group The experimental group was subcutaneously injected with 0.65 mg/mL Trimeresurus albolabris venom, 0.5 mL for each rat. The control group was subcutaneously injected with an equal amount of 0.9% physiological saline. Prior to and after injection, rats from these two groups were placed in the Morris Water Maze for recording of path length and escape latency. The remaining 60 rats were randomly allocated to another experimental group （n = 50） and another control group （n = 10）. Rats were correspondingly injected as described above. At different time points （0.1, 0.5, 1, 2, and 3 hours after injection）, rats were decapitated and bilateral hippocampal tissues were dissociated （approximately 100 mg for each sample）. Then, the acquired hippocampal tissue was immediately preserved at -70 ℃ for subsequent experiments. MAIN OUTCOME MEASURES： （1） The levels of histamine receptor （including H1R, H2R, and H3R） mRNA and protein in the hippocampi of rats were measured prior to and after injection of Trimeresurus albolabris venom using RT-PCR and Western Blot techniques. （2） Escape latency （namely, time to reach a platform） and path length were examined by Morris Water Maze testing. RESULTS： All 80 rats were included in the final analysis. In the experimental group, the level of mRNA for H3R receptor in rat hippocampi was just slightly changed, but the level of H3R receptor protein was significantly down-regulated compared with that in the control group （P 〈 0.05）. Both mRNA and protein levels for H1R receptor were initially downregulated and then recovered to normal levels. Expression of H2R receptor mRNA was initially upregulated, then downregulated, and finally restored to the control level. The level of H2R receptor protein showed a tendency for downregulation. In the Morris Water Maze testing, escape latency and path length were significantly longer in the experimental group than in the control group （P 〈 0.05）. CONCLUSION： Within three hours of injection with Trimeresurus albolabris venom, mRNA and protein levels of most histamine receptors in rat hippocampi were downregulated. Such changes possibly contribute to an impairment of memory and/or learning behaviors in rats following injection of Trimeresurus albolabris venom.

Purification and characterization of an arginine ester hydrolase from the venom of Trimeresurus mucrosqumatus in Hunan province of China

Objective: To study the physical and chemical properties of an arginine ester hydrolase from the venom of Trimeresurus mucrosqumatus in Hunan province of China.Methods:The arginine ester hydrolase(AEH) was isolated from the venom of Chinese Trimeresurus mucrosqumatus by a combination of ion-exchange chromatography on DEAE-Sephadex A-50, CM-Sepharose Cl-6B and gel filtration on Sephadex G-100.Results: The purified protein named TM-AEH,a glycoprotein with carbohydrate content of 0.5% neutral hexose and 0.75% sialic acid,a relative molecular mass of 29.0 kDa,and an isoelectric point(pI) of 5.2. It shares with an extinction coefficient(E 0.1%/cm) of 1.332 at 280 nm,consisted of 225 amino acid residues,and migrated as a band under reduced or non-reduced condition in basic PAGE.TM-AEH was a highly thermostable protein and was stable to pH changes between 5 and 9.The optimum temperature and optimum pH were 55℃ and 8.4 for its catalytic activity respectively,which was inhibited by Fe 3+ and Cu 2+.Conclusion:This protein can exhibit higher BAEE-hydrolysing activity and fibrinogenolytic activity as compared to that of whole venom.

Broad Antibacterial Activity of Bothrops jararaca Venom against Bacterial Clinical Isolates

Purpose: To evaluate the antibacterial activity of Bothrops jararaca venom against bacterial clinical isolates. Methods: Antibacterial activity of Bothrops jararaca venom was evaluated through agar diffusion method against the following bacteria: Acinetobacter baumannii, Oxacillinase-producing Acinetobacter baummanii, extended-spectrum β-lactamase-producing (ESBL) Enterobacter aerogenes, Escherichia coli, Escherichia coli ESBL, Klebsiella pneumoniae, Klebsiella pneumoniae ESBL, Proteus mirabilis, Pseudomonas aeruginosa, metallo β-lactamase-producing Pseudomonas aeruginosa, Staphylococcus aureus, oxacillin resistant Staphylococus aureus (ORSA), Staphylococcus epidermidis, and oxacillin resistant Staphylococus epidermidis.?Minimum inhibitory concentration was determined through microdilution plate protocol. Results: The venom presented antibacterial activity against all tested bacteria. More pronounced results were observed to Gram- positive bacteria, especially against ORSA. Conclusion: The present study evidenced the great antibacterial potential of Bothrops jararaca venom showing promising results even with resistant bacterial clinical isolates.

Animal venom studies: Current benefits and future developments

Poisonous organisms are represented in many taxa, including kingdom Animalia. During evolution, animals have developed special organs for production and injection of venoms. Animal venoms are complex mixtures, compositions of which depend on species producing venom. The most known and studied poisonous terrestrial animals are snakes, scorpions and spiders. Among marine animals, these are jellyfishes, anemones and cone snails. The toxic substances in the venom ofthese animals are mainly of protein and peptide origin. Recent studies have indicated that the single venom may contain up to several hundred different components producing diverse physiological effects. Bites or stings by certain poisonous species result in severe envenomations leading in some cases to death. This raises the problem of bite treatment. The most effective treatment so far is the application of antivenoms. To enhance the effectiveness of such treatments, the knowledge of venom composition is needed. On the other hand, venoms contain substances with unique biological properties, which can be used both in basic science and in clinical applications. The best example of toxin application in basic science is α-bungarotoxin the discovery of which made a big impact on the studies of nicotinic acetylcholine receptor. Today compositions of venom from many species have already been examined. Based on these data, one can conclude that venoms contain a large number of individual components belonging to a limited number of structural types. Often minor changes in the amino acid sequence give rise to new biological properties. Change in the living conditions of poisonous animals lead to alterations in the composition of venoms resulting in appearance of new toxins. At the same time introduction of new methods of proteomics and genomics lead to discoveries of new compounds, which may serve as research tools or as templates for the development of novel drugs. The application of these sensitive and comprehensive methods allows studying either of venoms available in tiny amounts or of low abundant components in already known venoms.

A life threatening scratch on little toe-at most clinical suspicion the essential key in management of snake bite

Snake bites are one among the under reported clinical emergencies from tropical countries.There are variations in clinical presentation of snake bites and its toxic features differ with the species and type of bite.There are lots of controversies in the treatment guidelines which often makes it difficult to manage.We report the case of a severe hemotoxic snake bite who presented to the outpatient service of our hospital with a trivial fool injury.Even though snakebites are familiar clinical situations for an emergency physician from tropics,we report this case as mast are under reported.We also intend to emphasize the excellent outcome of appropriately diagnosed and treated cases of snake bite.

Inhibition of Toxic Effects of Viper and Cobra Venom by Indian Medicinal Plants

The mortality rate from snakebites in West Bengal is very high and most of the deaths are caused by the Daboia russelli and Naja naja envenomation. Twenty-three plants from the seventeen families were collected from the traditional healers and explored for the first time for antisnake venom activity. In our previous report, the methanolic root extract of the Indian medicinal plants Pluchea indica, Hemidesmus indicus, Vitex negundo and Emblica officinalis significantly neutralized the Viper and Cobra venom-induced pathophysiological changes [1][2]. In the present study, we explored four plant extracts (Curcuma aromatica, Aristolochia indica, Androgrphis paniculata and Curcuma zeodaria) for the antisnake venom activity. The plant extracts significantly antagonized Daboia russelli, Echis carinatus, Ophiophagus hannah and Naja kaouthia venom-induced lethal activity both in in vitro and in vivo studies. Daboia russellii venom-induced haemorrhage, coagulant, defibrinogenating and PLA2 activity were significantly neutralized by the extracts. No precipitating bands were observed between the plant extract and venom. This observation confirmed the role of active constituents of plants and plant materials involved in snake venom inhibition. Further studies are going on in our laboratory for the identification of active molecules as well as their mechanism of venom inhibition.

Antitumor Activity of Disintegrin-Like Components from the Venom of <i>Montivipera</i><i>raddei</i>

Our findings represent the first report of the antitumor activity of the disintegrin-like components from the venom of Armenian viper (M. raddei). The venom of M. raddei was separated by reverse phase high-performance liquid chroma-tography (RP HPLC), and individual fractions were analyzed for disintegrin activity. Disintegrin-like components from the venom of M. raddei, by blocking integrins on breast cancer cells (MDA-MB-435), not only interferes with adhesion of breast cancer cells to the extracellular matrix, but also inhibits cellular mobility which is essential for cancer invasion. These effects seriously curtail the metastatic capability of the MDA-MB-435 cells.

A new fibrinogenase from Echis multisquamatis venom is a perspective agent for limited proteolysis and defibrinogenation

A serine proteinase with a fibrinogenase activity was isolated from the venom of viper Echis multisquamatis. Isolation was performed by the combination of Q-sepharose and Heparine-agarose chromatography. The enzyme has apparent molecular weight 35 ± 1 kDа. It posesses strong fibrinogen β-chain, moderate αchain proteolytic activity, arginine-amidase activity as the majority of serine fibrinogenases. The Km value was determined for β-chain fibrinogenolytic activity: Km = 8.3 μM. Kinetic parameters for amidase activity were also determined. Amino-acid composition was revealed. Limited hydrolysis of fibrinogen by the obtained fibrinogenase allowed us to detemine stable hydrolytic subproducts with definite molecular weights. The manner of the proteolytic processes suggests possible use of this fibrinogenase in probing fibrinogen structure dinamics by limited proteolysis. Applicability of the obtained fibrinogenase in therapeutic practice is speculative, but presented data about its nature are encouraging and require additional investigation.

毒蛇咬伤致多脏器损伤作用及机制研究进展

毒蛇咬伤是临床常见急重症,严重危害人类生命安全。蛇毒毒液螯入后会导致神经损伤、凝血障碍等致死性的全身性损伤,咬伤部位溃疡、肌坏死等致残性的局部损伤,以及多脏器组织损伤。近年来,毒蛇咬伤后血液毒性、神经毒性、细胞毒性被广泛研究,但对实体脏器的损伤研究较为缺乏,而蛇伤后多脏器损伤是其高致死率的重要原因。因此,该文对毒蛇咬伤后,合并致心脏、肝脏、肾脏、肺脏、脾脏、脑等实体脏器的功能性或器质性损伤作用及机制进行综述,旨在为蛇伤临床精准诊治提供参考与借鉴。