Enzymes of snake venoms

Snakes' venom is a mixture of biologically active substances, containing proteins and peptides. A number of these proteins interact with haemostasis system components. Activators and inhibitors affecting blood coagulation and fibrinolysis systems are of special interest. Venom components can be classified into three main groups, such as procoagulants, anticoagulants and fibrinolytic enzymes according to their action. This review is focused on enzymes from Agkistrodon halys halys venom. They are thrombine-like enzyme, named Ancystron-H, flbrinogenolytic enzyme, protein C activator and platelet aggregation inhibitor. Ancystron-H is used for determination of fibrinogen level in blood plasma of patients undergoing heparin treatment and blood coagulation inhibitors accumulation. The fibrinogenolytic enzyme can be used as the instrument for protein-protein interactions in fibrinogen-fibrin system. The protein C activator is used for protein C level determination in blood plasma with different pathologies. Functions of the platelet aggregation inhibitor, belonging to disintegrins group, can be used for development of antithrombotic preparations. Information about the use of snake venoms in science and medicine is presented.     

Mechanical behaviour of snake skin

Forty-nine samples of skin from the mid-ventral, ventrolateral and mid-dorsal regions of six species of snakes were mechanically tested in uniaxial extension at 0.05 strain/sec. The species of snakes studied ranged from generalists to specialists for arboreal or aquatic habitats. Analysis of the loading curves revealed substantial variation in loads and maximum stiffnesses among samples from different dorsoventral regions within an individual and among homologous samples from different species. Skin thickness varied by a factor of more than five, but this only partially accounts for the differences in the force per width of sample at a given extension. Qualitative differences in the dermal collagen fibres are implied by the shapes of the loading curves and the terminal elastic modulus which varied from about 15 to 585 MN/m². The strain at beginning of failure ranged from 0.12 to 0.60. The size of the scales within a skin sample was not a reliable predictor of the loading behaviour of the sample. Correlations between the mechanical behaviour of skin and specializations in locomotion and associated musculature are discussed.     

Electrophoretic analysis of snake venoms

Electrophoretic analyses were conducted on snake venoms from 21 species representing Elapidae, Crotalidae and Viperidae. Denatured and native venoms were analyzed by polyacrylamide gel electrophoretic (PAGE) methods with sodium dodecyl sulfate (SDS) and without SDS. Both SDS-PAGE and PAGE profiles of venoms from different snake species indicate that some proteins and polypeptide components of these venoms have common electrophoretic characteristics suggesting a genetic relationship. Conversely, the electropherograms also showed the characteristic protein and polypeptide profiles that could differentiate one snake species from another. Therefore, both SDS-PAGE and PAGE profiles suggest that proteins and polypeptides with similar characteristics abound among subspecies or related species, although each venom has a unique profile that differentiates one species from the other.     

Avidity of sera-neutralizing snake venom

Avidity of antivenom sera used for the treatment of snake bites was studied. Sera against the venom of Vipera libetina obtained from producers immunized with crude venoms were more avid than analogous sera obtained to anavenoms. In studying the avidity of polyvalent serum neutralizing the Vipera libetina, echis and cobra venoms showed the serum obtained in immunization with the mixture of crude venoms to be highly avid to all the venoms composing the antigen; besides, it bound the venoms of Vipera libetina and echis more rapidly and more stably than the corresponding monovalent sera.     

蛇神经毒素的研究进展

蛇毒是由许多种蛋白质、多肽、酶类以及其他小分子物质组成的混合物.在蛇毒中已经分离了许多种毒素分子,其中有一大类分子对哺乳动物的神经系统具有毒性效应,习惯上把这类分子成为蛇神经毒素.蛇神经毒素根据其作用位点的不同可以分为四大类:突触前蛇神经毒素、突触后蛇神经毒素、抗胆碱酯酶的蛇神经毒素和离子通道蛇神经毒素.许多蛇神经毒素已经分离纯化并进行了结构与功能的研究,几十近百种蛇神经毒素一级结构和空间结构已经得到测定.近几年来一些蛇神经毒素的基因文库以及cDNA文库已经构建出来,从中分离出的基因已经用于重组蛇神经毒素的生产研究.蛇神经毒素的分子结构与其功能具有较好的对应关系,即作用机制相同的毒素具有类似的空间结构.天然的蛇神经毒素以及重组的蛇神经毒素都已广泛应用于理论研究和一些临床应用.分离新的蛇神经毒素及其基因以及根据需要设计新的蛇神经毒素分子已成为该领域的热点,采用生物工程的方法规模生产蛇神经毒素也是当前及今后的研究方向.     

Adrenergic receptors of isolated snake atria

Cardiac adrenergic receptors in snakes were examined using an isolated atria preparation of Naja naja and Ptyas korros. Treatments included an examination of the atrial responses to selective alpha- and beta-adrenergic agonists and antagonists. In both species, both phenylephrine and isoproterenol produced dose-dependent increases in the atrial beating rate and tension. Phenylephrine-induced increases were characterized with a high affinity and low affinity components. These positive chronotropic and inotropic effects produced by phenylephrine and isoproterenol were abolished with propranolol and in the phenylephrine-induced response phentolamine also attenuated the low affinity response and blocked the high affinity response. With catecholamines depletion via 6-OH dopamine or reserpine, the high affinity component in the phenylephrine-induced response was no longer observed. It is concluded that beta-adrenoceptors are the predominant post-synaptic adrenoceptors in snake atria. Stimulatory presynaptic alpha-adrenoceptors for modulating noradrenaline release may also be present.     

Receptor variability-driven evolution of snake toxins

Three-finger toxins(TFTs) are well-recognized nonenzymatic venom proteins found in snakes. However,although TFTs exhibit accelerated evolution, the drivers of this evolution remain poorly understood.The structural complexes between long-chainα-neurotoxins, a subfamily of TFTs, and their nicotinic acetylcholine receptor targets have been determined in previous research, providing an opportunity to address such questions. In the current study, we observed several previously identified positively selected sites(PSSs) and the highly variable C-terminal loop of these toxins at the toxin/receptor interface. Of interest, analysis of the molecular adaptation of the toxin-recognition regions in the corresponding receptors provided no statistical evidence for positive selection. However, these regions accumulated abundant amino acid variations in the receptors from the prey of snakes, suggesting that accelerated substitution of TFTs could be a consequence of adaptation to these variations. To the best of our knowledge, this atypical evolution, initially discovered in scorpions, is reported in snake toxins for the first time and may be applicable for the evolution of toxins from other venomous animals.     

Intrinsic organization of snake lateral cortex

Lateral cortex is the most laterally placed of the four cortical areas in snakes. Earlier studies suggest that it is composed of several subdivisions but provide no information on their organization. This paper first investigates the structure of lateral cortex in boa constrictors (Constrictor constrictor), garter snakes (Thamnophis sirtalis), and banded water snakes (Natrix sipedon) using Nissl and Golgi preparations; and secondly examines the relation of main olfactory bulb projections to the subdivisions of lateral cortex using Fink-Heimer and electron microscopic preparations. Lateral cortex is divided on cytoarchitectonic grounds into two major parts called rostral and caudal lateral cortex. Each part is further divided into dorsal and ventral subdivisions so that lateral cortex has a total of four subdivisions: dorsal rostral lateral cortex (drL), ventral rostral lateral cortex (vrL), dorsal caudal lateral cortex (dcL) and ventral caudal lateral cortex (vcL). Systematic analyses of Golgi preparations indicate that the rostral and caudal parts each contain distinct populations of neurons. Rostral lateral cortex contains bowl cells whose dendrites arborize widely in the outer cortical layer (layer 1). The axons of some bowl cells can be traced medially into dorsal cortex, dorsomedial cortex and medial cortex. Caudal lateral cortex contains pyramidal cells whose somata occur in layers 2 and 3 and whose dendrites extend radially up to the pial surface. In addition, three populations of neurons occur in both rostral and caudal lateral cortex. Stellate cells occur in all three layers and have dendrites which arborize in all directions. Double pyramidal cells occur primarily in layer 2 and have dendrites which form two conical fields whose long axes are oriented radially. Horizontal cells occur in layer 3 and have dendrites oriented concentric with the ependyma. Fink-Heimer preparations of snakes which underwent lesions of the main olfactory bulb show that the primary olfactory projections to cortex are bilateral and restricted precisely to rostral lateral cortex. Electron microscopic degeneration experiments indicate that the olfactory bulb fibers end as terminals which have clear, spherical vesicles and asymmetric active zones. The majority are presynaptic to dendritic spines in outer layer 1. These studies establish that lateral cortex in snakes is heterogeneous and contains two major parts, each containing two subdivisions. The rostral and caudal parts have characteristic neuronal populations. Primary olfactory input is restricted to rostral lateral cortex and seems to terminate heavily on the distal dendrites of bowl cells. Axons of some of these cells leave lateral cortex, so that the rostral lateral cortex forms a direct route by which olfactory information reaches other cortical areas. The functional role of caudal lateral cortex is not clear.     

Mitogenic activity of snake venom lectins

Five lactose-inhibitable lectins have been isolated from snake venoms. These five share certain biochemical properties but are not identical (Gartner, Stocker & Williams, 1980; Gartner & Ogilvie, 1984). In this study the lectins were tested for their ability to stimulate lymphocytes to undergo DNA synthesis. We found that three of the lectins were comparable in mitogenic activity to the T cell lectin, concanavalin A (Con A). The mitogenic activity was blocked by lactose, a sugar which also blocks the haemagglutination activity of these lectins. Although mitogenic response appeared to be due to T cells, it depended on the presence of accessory cells in the culture. This requirement for macrophages could be replaced by the phorbol ester tumour promoter, 12-o-tetradecanoylphorbol-13-acetate (TPA).     

Molecular evolution of snake venom toxins

Summary Phylogenetic trees were constructed for 62 venom toxins of snakes ofProteroglyphae suborder using matrix method. The resulting tree fromMinimum Spanning Tree-Cluster Analysis technique had the lowest percent deviation (8.55). The taxonomic relationship of these toxins agrees very well with zoological opinions. However, the appearance of the tree did not directly provide a plausible evolutionary model for the toxins. A model was derived from nodal ancestral sequence calculations, comparisons between intra-and inter-generical rates of amino acid change, and generally held ideas about protein evolution. According to the model, short neurotoxin is the ancient form of snake venom toxins. The courses of evolution leading to the present intraspecific homologous toxins are explained by gene duplication and allelomorphism.     

Inflammatory effects of snake venom metalloproteinases

Metalloproteinases are abundant enzymes in crotaline and viperine snake venoms. They are relevant in the pathophysiology of envenomation, being responsible for local and systemic hemorrhage frequently observed in the victims. Snake venom metalloproteinases (SVMP) are zinc-dependent enzymes of varying molecular weights having multidomain organization. Some SVMP comprise only the proteinase domain, whereas others also contain a disintegrin-like domain, cysteine-rich, and lectin domains. They have strong structural similarities with both mammalian matrix metalloproteinases (MMP) and members of ADAMs (a disintegrin and metalloproteinase) group. Besides hemorrhage, snake venom metalloproteinase induce local myonecrosis, skin damage, and inflammatory reaction in experimental models. Local inflammation is an important characteristic of snakebite envenomations inflicted by viperine and crotaline snake species. Thus, in the recent years there is a growing effort to understand the mechanisms responsible for SVMP-induced inflammatory reaction and the structural determinants of this effect. This short review focuses the inflammatory effects evoked by SVMP.