Purification,Characterization, and Chemical Modification of Neurotoxic Peptide from Daboia russelii Snake Venom of India

Comprehensive knowledge of venom composition is very important for effective management of snake envenomation and antivenom preparation. Daboia russelii venom from the eastern region of India is the most neurotoxic among the four venom samples investigated. From the eastern D. russelii venom sample, neurotoxic peptide has been purified by combined method of ion exchange gel permeation chromatography and reversed phase high performance liquid chromatography. Molecular weight of Daboia neurotoxin III (DNTx‐III) found to be 6,849 Da (as measured on matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometer), and N‐terminal amino acid sequences is I K C F I T P D U T S Q A. Approximate LD₅₀ dosage was 0.24 mg/kg body weight. It produced concentration‐ and time‐dependent inhibition of indirectly stimulated twitches of Rana hexadactyla sciatic nerve gastrocnemius muscle preparations. Chemical modification of DNTx‐III tryptophan residue(s) reduced the twitch height inhibition property of toxin, signifying the importance of tryptophan residues for the neurotoxic function. This type of neurotoxic peptide is unique to east Indian regional D. russelii venom. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:295‐304, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21486     

绿瘦蛇捕食鳄蜥的现象

鳄蜥(Shinisauruscrocodilurus)是濒危的孑遗物种,生存遭受多方威胁。除人为猎杀和生境受到破坏之外,天敌也是其致命的威胁之一,但目前关于其被天敌捕食的研究非常匮乏。本文报道了绿瘦蛇(Ahaetullaprasina)捕食鳄蜥的现象,以全事件记录法观察了这一行为过程。鳄蜥面对的被捕食风险可能较广。除蛇外,鳄蜥的天敌也可能是鸟类等动物。在饲养繁育中,要注意加强防范举措,以免鳄蜥被天敌侵害。     

Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

A novel short neurotoxin,cobrotoxin c,from monocellate cobra (Naja kaouthia) venom: isolation and purification,primary and secondary structure determination,and tertiary structure modeling

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR).     

Metalloproteinase with factor X activating and fibrinogenolytic activities from Vipera berus berus venom

We have previously shown that Vipera berus berus venom contains several factor X activating enzymes. In the present study we have investigated one of them. The enzyme was separated from venom by gel filtration on Sephadex G-100 superfine and chromatography on agarose HPS-7 and phenyl-agarose. The enzyme is a glycosylated metalloproteinase containing hexoses, hexosamines and neuraminic acid. The purified factor X activating enzyme consists of two equal chains (59 kDa). The specificity studies have shown that enzyme is nonspecific factor X activating proteinase hydrolysing also proteins such as azocasein, gelatin and fibrinogen. The enzyme hydrolyses oxidized insulin B-chain at the positions Ala¹⁴–Leu¹⁵ and Tyr¹⁶–Leu¹⁷ but it is inactive on fibrin, plasminogen and prothrombin. We used 8–10 amino acid residues containing peptides, which reproduce the sequence around the cleavage sites in factor X, factor IX and fibrinogen, as potential substrates for enzyme. Cleavage products of peptide hydrolysis were determined by MALDI-TOF MS. The peptide Asn–Asn–Leu–Thr–Arg–Ile–Val–Gly–Gly—factor X fragment was cleaved by enzyme at positions Leu³–Thr⁴ and Arg⁵–Ile⁶. The fibrinogen peptide fragment Glu–Tyr–His–Thr–Glu–Lys–Leu–Val–Thr–Ser was hydrolysed at position Lys⁶–Leu⁷.     

Distribution of lipids from the yolk to the tissues during development of the water python (<Emphasis Type="Italic">Liasis fuscus</Emphasis>)

Energy metabolism during embryonic development of snakes differs in several respects from the patterns displayed by other reptiles. There are, however, no previous reports describing the main energy source for development, the yolk lipids, in snake eggs. There is also no information on the distribution of yolk fatty acids to the tissues during snake development. In eggs of the water python (Liasis fuscus), we report that triacylglycerol, phospholipid, cholesteryl ester and free cholesterol, respectively, form 70.3%, 14.1%, 5.7% and 2.1% of the total lipid. The main polyunsaturate of the yolk lipid classes is 18:2n-6. The yolk phospholipid contains 20:4n-6 and 22:6n-3 at 13.0% and 3.6% (w/w), respectively. Approximately 10% and 30% of the initial egg lipids are respectively recovered in the residual yolk and the fat body of the hatchling. A major function of yolk lipid is, therefore, to provision the neonate with large energy reserves. The proportion of 22:6n-3 in brain phospholipid of the hatchling is 11.1% (w/w): this represents only 0.24% of the amount of 22:6n-3 originally present in the egg. This also contrasts with values for free-living avian species where the proportion of DHA in neonatal brain phospholipid is 16–19%. In the liver of the newly hatched python, triacylglycerol, phospholipid and cholesteryl ester, respectively, form 68.2%, 7.7% and 14.3% of total lipid. This contrasts with embryos of birds where cholesteryl ester forms up to 80% of total liver lipid and suggests that the mechanism of lipid transfer in the water python embryo differs in some respects from the avian situation.Abbreviations ARA arachidonic acid - DHA docosahexaenoic acidCommunicated by G. Heldmaier     

内蒙古蛇类新纪录——双斑锦蛇

2007年8月17日在内蒙古赤峰市巴林右旗北部的赛罕乌拉国家级自然保护区进行野生动物多样性调查时,首次发现双斑锦蛇(Elaphe bimaculata),随后又分别于2008年8月、2009年10月4次在保护区内发现该蛇种,2次为成年个体,1次为幼体,经核查鉴定该蛇为内蒙古蛇类新纪录。     

Snake venoms as a source of compounds modulating sperm physiology: Secreted phospholipases A2 from Oxyuranus scutellatus scutellatus impact sperm motility,acrosome reaction and in vitro fertilization in mice

The goal of this study was to identify new compounds from venoms able to modulate sperm physiology and more particularly sperm motility. For this purpose, we screened the effects of 16 snake venoms cleared of molecules higher than 15 kDa on sperm motility. Venoms rich in neurotoxins like those from Oxyuranus scutellatus scutellatus or Daboia russelii, were highly potent inhibitors of sperm motility. In contrast, venoms rich in myotoxins like those from Echis carinatus, Bothrops alternatus and Macrovipera lebetina, were inactive. From the main pharmacologically-active fraction of the Taipan snake O. scutellatus s., a proteomic approach allowed us to identify 16 different proteins, among which OS1 and OS2, two secreted phospholipases A2 (sPLA₂). Purified OS1 and OS2 mimicked the inhibitory effect on sperm motility and were likely responsible for the inhibitory effect of the active fraction. OS1 and OS2 triggered sperm acrosome reaction and induced lipid rearrangements of the plasma membrane. The catalytic activity of OS2 was required to modulate sperm physiology since catalytically inactive mutants had no effect. Finally, sperm treated with OS2 were less competent than control sperm to initiate in vitro normal embryo development. This is the first report characterizing sPLA₂ toxins that modulate in vitro sperm physiology.     

Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2

Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A₂ inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A₂ (PLA₂s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA₂ contained in this venom. Furthermore, these effects of acidic PLA₂ on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver.