Post-translational modification accounts for the presence of varied forms of nerve growth factor in Australian elapid snake venoms

The Australian elapid snakes are amongst the most venomous snakes in the world, but much less is known about the overall venom composition in comparison to Asian and American snakes. We have used a combined approach of cDNA cloning and 2-DE with MS to identify nerve growth factor (NGF) in venoms of the Australian elapid snakes and demonstrate its neurite outgrowth activity While a single 730 nucleotide ORF, coding for a 243 amino acid precursor protein was detected in all snakes, use of 2-DE identified NGF proteins with considerable variation in molecular size within and between the different snakes. The variation in size can be explained at least in part by Winked glycosylation. it is possible that these modifications alter the stability, is necessary to activity and other characteristics of the snake NGFs. Further characterisation delineate the function of the individual NGF isoforms.

Fascinating and forgotten, the conservation status of marine elapid snakes

An assessment of marine elapid snakes found 9% of marine elapids are threatened with extinction, and an additional 6% are Near Threatened. A large portion (34%) is Data Deficient. An analysis of distributions revealed the greatest species diversity is found in Southeast Asia and northern Australia. Three of the seven threatened species occur at Ashmore and Hibernia Reefs in the Timor Sea, while the remaining threatened taxa occur in the Philippines, Niue, and Solomon Islands. The majority of Data Deficient species are found in Southeast Asia. Threats to marine snakes include loss of coral reefs and coastal habitat, incidental bycatch in fisheries, as well as fisheries that target snakes for leather. The presence of two Critically Endangered and one Endangered species in the Timor Sea suggests the area is of particular conservation concern. More rigorous, long-term monitoring of populations is needed to evaluate the success of "conservation measures" for marine snake species, provide scientifically based guidance for determining harvest quotas, and to assess the populations of many Data Deficient species.

Identification and characterization of novel reptile cathelicidins from elapid snakes

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576 bp an

Reproductive ecology of dipsadine snakes, with emphasis on South American species

A relatively large amount of variation occurs in the reproductive ecology of tropical snakes, and this variation is generally regarded as being a consequence of seasonality in climate and prey availability. In some groups, even closely related species may differ in their reproductive ecology; however, in others it seems to be very conservative. Here we explore whether characters related to reproduction are phylogenetically constrained in a monophyletic group of snakes, the subfamily Dipsadinae, which ranges from Mexico to southern South America. We provide original data on reproduction for Leptodeira annulata, Imantodes cenchoa, and three species of Sibynomorphus from southern, southeastern and central Brazil, and data from literature for other species and populations of dipsadines. Follicular cycles were seasonal in Atractus reticulatus, Dipsa, albifrons, Hypsiglena torquata, Leptodeira maculata, L. punctata, Sibynomorphus spp. and Sibon sanniola from areas where climate is seasonal. In contrast, extended or continuous follicular cycles were recorded in Dipsas catesbyi, D. neivai, Imantodes cenchoa, Leptodeira annulata, and Ninia maculata from areas with seasonal and aseasonal climates. Testicular cycles also varied from seasonal (in H. torquiata) to continuous (in Dipsa,5 spp., Leptodeira annulata, L. maculata, N. maculata, and Sibynomorphus spp.). Most dipsadines are small (less than 500 rum SVL), and females attain sexual maturity with similar relative body size than males. Sexual dimorphism occurred in terms of SVL and tail length in most species, and clutch size tended to be small (less than five eggs). Combat behavior occurs in Imantodes cenchoa, which did not show sexual size dimorphism. Reproductive timing, for both females and males, varied among species but in general there were no differences between the tribes of Dipsadinae in most of the reproductive characteristics, such as mean body size, relative size at sexual maturity, sexual size and tail dimorphism, duration of vitellogenesis or egg-carrying in oviducts.

Comparative analysis of prothrombin activators from the venom of Australian elapids

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

Cloning and characterization of natriuretic peptides from the venom glands of Australian elapids

The venom from Australian elapid snakes contains a complex mixture of polypeptide toxins that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Included in these toxin families are the recently described venom natriuretic peptides, which display similar structure and vasoactive functions to mammalian natriuretic peptides. This paper describes the identification and detailed comparative analysis of the cDNA transcripts coding for the mature natriuretic peptide from a total of nine Australian elapid snake species. Multiple isoforms were identified in a number of species and represent the first description of a natriuretic peptide from the venom gland for most of these snakes. Two distinct natriuretic peptide isoforms were selected from the common brown snake (Pseudonaja textilis), PtNP-a, and the mulga (Pseudechis australis), PaNP-c, for recombinant protein expression and functional analysis. Only one of these peptides, PtNP-a, displayed cGMP stimulation indicative of normal natriuretic peptide activity. Interestingly, both recombinant peptides demonstrated a dose-dependent inhibition of angiotensin converting enzyme (ACE) activity, which is predictive of the vasoactive effects of the toxin. The natriuretic peptides, however, did not possess any coagulopathic activity, nor did they inhibit or potentiate thrombin, adenosine diphosphate or arachidonic acid induced platelet aggregation. The data presented in this study represent a significant resource for understanding the role of various natriuretic peptides isoforms during the envenomation process by Australian elapid snakes. (c) 2006 Published by Elsevier Masson SAS.

Life History Aspects of Oxyrhopus trigeminus (Serpentes: Dipsadidae) from Two Sites in Southeastern Brazil

We studied the reproduction, sexual dimorphism, and diet of Oxyrhopus trigeminus from two sites in southeastern Brazil. Oxyrhopus trigeminus from Irape Power Plant (IPP) contained vitellogenic follicles and eggs in both rainy and dry seasons and clutch size was not correlated with female snout vent length (SVL). Sexual dimorphism was evident. Females attain larger SVL but males have longer tails. We found three females from Santa Clara Power Plant (SPP) with vitellogenic follicles, all of them collected in the dry season. Mean SVLs of adult females from IPP and SPP were 717.7 mm and 786 mm, respectively. Mean SVL of adult males from IPP was 553.4 mm and the single adult male from SPP was 507 mm. The diet of O. trigeminus from IPP included rodents (46.7%), lizards (33.3%), and birds (20%). The volume of individual prey items was not correlated with snake SVL. The diet of O. trigeminus from SPP included rodents (37.5%), lizards (37.5%), birds (12.5%), and marsupials (12.5%). It seemed that an ontogenetic shift may occur in individuals of this snake species from IPP.

Identification and analysis of venom gland-specific genes from the coastal taipan (Oxyuranus scutellatus) and related species

Australian terrestrial elapid snakes contain amongst the most potently toxic venoms known. However, despite the well-documented clinical effects of snake bite, little research has focussed on individual venom components at the molecular level. To further characterise the components of Australian elapid venoms, a complementary (cDNA) microarray was produced from the venom gland of the coastal taipan (Oxyuranus scutellatus) and subsequently screened for venom gland-specific transcripts. A number of putative toxin genes were identified, including neurotoxins, phospholipases, a pseudechetoxin-like gene, a venom natriuretic peptide and a nerve growth factor together with other genes involved in cellular maintenance. Venom gland-specific components also included a calglandulin-like protein implicated in the secretion of toxins from the gland into the venom. These toxin transcripts were subsequently identified in seven other related snake species, producing a detailed comparative analysis at the cDNA and protein levels. This study represents the most detailed description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.

Molecular diversity in venom from the Australian Brown Snake, Pseudonaja textilis

Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A(2)s, and pre-and postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two- dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, gamma-carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents.

Toxinology in Australia's colonial era: A chronology and perspective of human envenomation in 19th century Australia

The medical management of those envenomed by snakes, spiders and poisonous fish in Australia featured extensively in the writings 19th century doctors, expeditioners and anthropologists. Against the background of this introduced medical doctrine there already existed an extensive tradition of Aboriginal medical lore; techniques of heat treatment, suction, incision and the application of plant-derived pharmacological substances featured extensively in the management of envenomed victims. The application of a hair-string or grass-string ligature, suctioning of the bite-site and incision were practised in a variety of combinations. Such evolved independently of and pre-dated such practices, which were promoted extensively by immigrant European doctors in the late 19th century. Pacific scientific toxinology began in the 17th century with Don Diego de Prado y Tovar's 1606 account of ciguatera. By the end of the 19th century more than 30 papers and books had defined the natural history of Australian elapid poisoning. The medical management of snakebite in Australia was the focus of great controversy from 1860 to 1900. Dogmatic claims of the supposed antidote efficacy of intravenous ammonia by Professor G.B. Halford, and that of strychnine by Dr. Augustus Mueller, claimed mainstream medical attention. This era of potential iatrogenic disaster and dogma was brought to a conclusion by the objective experiments of Joseph Lauterer and Thomas Lane Bancroft in 1890 in Brisbane; and by those of C.J. Martin (from 1893) and Frank Tidswell (from 1898), both of Sydney. The modern era of Australian toxinology developed as a direct consequence of Calmette's discovery, in Paris in 1894, of immune serum, which was protective against snakebite. We review the key contributors and discoveries of toxinology in colonial Australia.

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.

Elapid Snake Venom Analyses Show the Specificity of the Peptide Composition at the Level of Genera Naja and Notechis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)