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.

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.

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

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.

Effects of Batroxobin on Spatial Learning and Memory Disorder of Rats with Temporal Ischemia and the Expression of HSP32 and HSP70

The effect of Batroxobin on spatial memory disorder of left temporal ischemic rats and the expression of HSP32 and HSP70 were investigated with Morri`s water maze and immunohistochemistry methods. The results showed that the mean reaction time and distance of temporal ischemic rats in searching a goal were significantly longer than those of the sham-operated rats and at the same time HSP32 and HSP70 expression of left temporal ischemic region in rats was significantly increased as compared with the sham-operated rats. However, the mean reaction time and distance of the Batroxobin-treated rats were shorter and they used normal strategies more often and earlier than those of ischemic rats. The number of HSP32 and HSP70 immune reactive cells of Batroxobin-treated rats was also less than that of the ischemic group. In conclusion, Batroxobin can improve spatial memory disorder of temporal ischemic rats; and the down-regulation of the expression of HSP32 and HSP70 is probably related to the attenuation of ischemic injury.

Essential Oils from Mentha piperita,Cymbopogon citratus,Rosmarinus officinalis,Peumus boldus and Foeniculum vulgare:Inhibition of Phospholipase A_(2) and Cytotoxicity to Human Erythrocytes

The essential oils from Mentha piperita, Cymbopogon citratus, Rosmarinus officinalis, Peumus boldus and Foeniculum vulgare were extracted by hydrodistillation and characterized and quantified by GC-MS and GC-DIC. The oils induced hemolysis with all the doses evaluated (0.6 to 1.8 μL), and the diameters of the halos varied between 9 and 15 mm. Pre-incubation of P. boldus oil with Bothrops jararacussu venom resulted in potentiation of venom-induced hemolysis (30%) (proteases and phospholipases A2). The essential oil from M. piperita (0.6 μL) inhibited venom-induced hemolysis by 45%, whereas 0.6 μL of R. officinalis oil increased the hemolysis by 20%. For the essential oil from F. vulgare, 100% inhibition of activity (0.6 and 1.2 μL) was observed. The application of C. citratus oil induced hemolysis with all the volumes evaluated. Phospholipase activity induced by the venom was only inhibited (10%) with the 0.6 μL volume of R. officinalis oil. The oils from M. piperita and F. vulgare (1.8 μL) and C. citratus oil (0.6 μL) potentiated the phospholipase activity. The results highlight the need for a broad characterization and regulation of the use of natural products, because they can have therapeutic or toxic actions.

Systematics and Species Validity of the Dabieshan Pit Viper Protobothrops dabieshanensis Huang et al. 2012: Evidence from a Mitochondrial Gene Sequence Analysis

Aimed to evaluate the phylogenetic position of the recently described Protobothrops dabieshanensis Huang et al. （2012）, phylogenic relationships of 12 species within Protobothrops based on four mtDNA gene fragments （12S RNA, 16S RNA, ND4 and Cyt b） were reconstructed in our study. The result indicates a clade composed ofP dabiesha- nensis, P. jerdonii and P xiangchengsis with strong support. The genetic distance among P dabieshanensis, P jerdonii and P xiangchengsis was much lower than other congeners. Based on the data from the phylogenetic analysis and pre- viously described morphological differences, we conclude that P dabieshanensis is a valid species with close affinities to P jerdonii and P xiangchengsis.

Molecular Phylogeny of the Genus Gloydius(Serpentes: Crotalinae)

Based on two mitochondrial genes (cyt b, ND4) and one nuclear gene (c-mos), we explored the relationships within the Asian pit viper genus Gloydius. In total, 23 samples representing 10 species were analyzed. All phylogenetic analyses support a monophyletic Gloydius with two major clades, one comprising G. brevicaudus, G. blomhoffii, and G. ussuriensis with the sister clade consisting of G. intermedius, G. saxatilis, G. halys and G. shedaoensis. The relationships among the three montane species G. strauchi, G. qinlingensis and G. liupanensis, as well as the two monophyletic groups, are unstable, and discussed. Divergence date estimation indicates that Gloydius lineage formed 15 Ma and diversification of the genus occurred at 9.89 Ma. Issues regarding the taxonomy of this genus are discussed where necessary.

Sexual Dimorphism and Geographic Variation in the Asian Lance-headed Pitviper Protobothrops mucrosquamatus in the China's Mainland

Sexual dimorphism（SD） and geographic variation（GV） are widespread in snakes. Protobothrops mucrosquamatus（Cantor 1839） is one of the most common Asian venomous snakes with a wide geographical distribution. We examined SD and GV patterns for this species by using multivariate statistical analyses of external morphological characters scored from specimens from the China's Mainland. The result displayed that SD was significant in several external characters in P. mucrosquamatus, and the male P. mucrosquamatus formed two distinct clusters（Hainan Island and China's Mainland）, but the females did not. Based on our present work and the other data, we concluded that no significantly intraspecific differentiation is present within this species.

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.

SCREENING A CDNA LIBRARY FOR PHOSPHOLIPASE A2 CLONES USING BLOOD AGAR PLATES

A technique was developed for detecting venom phospholipase A2(PLA2) using serum and red blood cells. M1 and M2, two PLA2s isolated from Crotalus m.molossus (Northern black-tail rattlesnake), were used for preliminary development of the as say. Various combinations of human, sheep, rat, and mouse red blood cells (RBC) with human,rat,and mouse sera were tested on their effectiveness to detect PLA2.Complete hemolysis (b hemolysis) was evident in the plate with rat RBC mixed with mouse serum.No hemolysis was detected in plates containing human RBC and human serum. Human RBC mixed with mouse serum proved to be ideal, even though this combination displayed incomplete hemolysis(a hemolysis).Susceptible RBC, in conjunction with rat or mouse serum, are excellent indicators for the presence of PLA2.Mixtures of RBC and serum in combination with BB4(E. coli) cells,λbacteriophage,and IPTG on LB agar plates provide an excellent detection system for cDNA clones that express venonl PLA2. Hemolysis surrounding a plaque is identified as positive for PLA2.

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.

In vitro antibacterial activity of venom protein isolated from sea snake Enhydrina schistosa against drug-resistant human pathogenic bacterial strains

Objective:To evaluate the antibacterial activity of sea snake(Enhydrina schistosa)venom protein against drug-resistant human pathogenic bacterial strains.Methods:The venom was collected by milking process from the live specimens of sea snake are using capillary tubes or glass plates.Venom was purified by ion exchange chromatography and it was tested for in-vitro antibacterial activity against 10 drug-resistant human pathogenic bacterial strains using the standard disc diffusion method.Results:The notable antibacterial activity was observed at 150μg/mL concentration of purified venom and gave its minimum inhibitory concentrations values exhibited between 200-100μg/mL against all the tested bacterial strains.The maximum zone of inhibition was observed at 16.4 mm against Salmonella boydii and the minimum activity was observed at 7.5 mm against Pseudomonas aeruginosa.After the sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis there were a clear single band was detected in the gel that corresponding to purified venom protein molecular weight of 44 kDa.Conclusions:These results suggested that the sea snake venom might be a feasible source for searching potential antibiotics agents against human pathogenic diseases.

Allergic reactions to antivenom in a patient bitten twice by the same snake within a month： A rare case report and literature review

Antivenom is the most effective method currently available for the treatment of poisonous snake bite. Allergic reactions to antivenom have been reported in the past. Here we shared a case of allergic re- actions to antivenom in an old male patient who was bitten twice by the same snake （probably same one） at the same biting site within a month whereas the patient did not show any allergic disorder in the first bitten. Envenomations twice in a short period time by the same kind of snake are very rare. Physician should be alert to the occurrence of allergic reactions in treating this type of patients with antivenom. The skin allergy test has a certain value in predicting the allergic response before the second use of antivenom. Desensitization may reduce the incidence of allergic reactions, but this is insufficient. Rather than non-lgE-mediated immediate hypersensitivity, patients receiving the second treatment of antivenom may develop IgE-mediated immediate hypersensitivity. Once happened, the antivenom treatment should be stopped promptly and anti-allergy treatment should be given immediately.

Snakes that Snack on Poison Predators take venom from prey and use it themselves

动物学家发现,虎斑颈槽蛇通过吃一种分泌二烯羟酸内脂毒素的蟾蜍将毒素储存在身体内以抵御捕食者的危害。它会将毒素储存在颈背部,当遇到天敌鹰时通常拱起颈部,如受到威胁便会释放储存的毒素。但目前专家还没有研究出到底虎斑颈槽蛇是如何在不消化毒素的前提下将毒素储存起来并转移到毒腺里的,而毒素在其体内又经过了怎样的化学转变。

Efficacy of Silene arenosa extract on acetylcholinesterase in Bungarus sindanus(krait) venom

OBJECTIVE:To examine the efficacy of Silene arenosa extract on acetylcholinesterase (AChE) of krait (Bungarus Sindanus) snake venom.METHODS:The present project designed to evaluate the inhibition of AChE by following standard procedures.RESULTS:Statistical analysis of the results showed that Silene arenosa exerted 73%inhibition against the krait venom acetylcholinesterase at fixed substrate acetylcholine (ACh) concentration (0.5 mM).Kinetic analysis using the Lineweaver Burk plot revealed that Silene arenosa caused a competitive type of inhibition i.e.K_(m) values increased from 26.6 to 93.3 mM (26.6%to 93.3%) and V_(max)remained constant in a concentration-dependent manner.Silene arenosa competes with the substrate to bind at the active site of the enzyme.The K_(mapp)of venom AChE for Silene arenosa increased from 60%to 81.6%and the V_(maxapp)remains constant.K_(i)(inhibition constant was estimated to be 48μg for snake venom);while the K_(m)(Michaelis-Menten constant of AChE-substrate into AChE and product) was estimated to be 0.5 mM.The IC_(50)of AchE calculated for Silene arenosa was 67μg.CONCLUSION:The present results suggest that Silene arenosa extract can be considered as an inhibitor of snake venom AChE.