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.

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MITI, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pia ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 mu M, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors. (c) 2005 Elsevier Inc. All rights reserved.

Novel natriuretic peptides from the venom of the inland taipan (Oxyuranus microlepidotus): isolation, chemical and biological characterisation

Three natriuretic-like peptides (TNP-a, TNP-b, and TNP-c) were isolated from the venom of Oxyuranus microlepidotus (inland taipan) and were also present in the venoms of Oxyuranus scutellatus canni (New Guinea taipan) and Oxyuranus scutellatus scutellatus (coastal taipan). They were isolated by HPLC, characterised by mass spectrometry and Edman analysis, and consist of 35-39 amino acid residues. These molecules differ from ANP/BNP through replacement of invariant residues within the 17-membered ring structure and by inclusion of proline residues in the C-terminal tail. TNP-c was equipotent to ANP in specific GC-A assays or aortic ring assays whereas TNP-a and TNP-b were either inactive (GC-A over-expressing cells and endothelium-denuded aortic rings) or weakly active (endothelium-in tact aortic rings). TNP-a and TNP-b were also unable to competitively inhibit the binding of TNP-c in endothelium-denuded aortae (GC-A) or endothelium-in tact aortae (NPR-C). Thus, these naturally occurring isoforms provide a new platform for further investigation of structure-function relationships of natriuretic peptides. (C) 2004 Elsevier Inc. All rights reserved.

The Thelastomatoidea (Nematoda : Oxyurida) of two sympatric Panesthiinae (Insecta : Blattodea) from southeastern Queensland, Australia: taxonomy, species richness and host specificity

The thelastomatoid fauna of two species of wood-burrowing cockroach (Blattodea, Blaberidae), Panesthia cribrata and Panesthia tryoni tryoni, from Lamington National Park, Australia, is described. The following eight new species and three new genera of thelastomatid are proposed: Bilobostoma exerovulva n. g., n. sp.; Cordonicola gibsoni n. sp.; Coronostoma australiae n. sp.; Desmicola ornata n. sp.; Hammerschmidtiella hochi n. sp.; Malaspinanema goateri n. g., n. sp.; Travassosinema jaidenae n. sp.; and Tsuganema cribratum n. g., n. sp. Additional data are given for Blattophila sphaerolaima and Leidynemella fusiformis. Of the 11 species reported, nine were found in P. cribrata and ten in P. tryoni tryoni. Such levels of thelastomatoid species richnessness in single host species are exceptional. Only the mole cricket, Gryllotalpa africana (23), and the domestic cockroach, Periplaneta americana (20), have higher reported richness. Three species, T jaidenae, C. australiae and D. ornata, were found either exclusively or significantly more prevalently in P tryoni tryoni than in R cribrata. Species of Travassosinema, Coronostoma and Desmicola have been found previously only in millipedes (Diplopoda), a fact that suggests that there is a greater degree of niche overlap between R tryoni tryoni and millipedes than for R cribrata.

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.

An examination of the dynamic relationship between self-efficacy and performance across levels of analysis and levels of specificity

This research used resource allocation theory to generate predictions regarding dynamic relationships between self-efficacy and task performance from 2 levels of analysis and specificity. Participants were given multiple trials of practice on an air traffic control task. Measures of task-specific self-efficacy and performance were taken at repeated intervals. The authors used multilevel analysis to demonstrate differential and dynamic effects. As predicted, task-specific self-efficacy was negatively associated with task performance at the within-person level. On the other hand, average levels of task-specific self-efficacy were positively related to performance at the between-persons level and mediated the effect of general self-efficacy. The key findings from this research relate to dynamic effects - these results show that self-efficacy effects can change over time, but it depends on the level of analysis and specificity at which self-efficacy is conceptualized. These novel findings emphasize the importance of conceptualizing self-efficacy within a multilevel and multispecificity framework and make a significant contribution to understanding the way this construct relates to task performance.

Cyclic MrIA: A stable and potent cyclic conotoxin with a novel topological fold that targets the norepinephrine transporter

Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the chi-conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the, beta-hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET. Furthermore, the cyclic version has increased resistance to trypsin digestion relative to the native peptide, an intriguing result because the cleavage site for the trypsin is not close to the cyclization site. The use of peptides as drugs is generally hampered by susceptibility to proteolysis, and so, the increase in enzymatic stability against trypsin observed in the current study may be useful in improving the therapeutic potential of MrIA. Furthermore, the structure reported here for cyclic MrIA represents a new topology among a growing number of circular disulfide-rich peptides.

Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a D-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Mammalian L-to-D-amino-acid-residue isomerase from platypus venom

The presence Of D-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian D-amino-acid-residue isomerase(s) responsible for the modification of the all-L-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is similar to 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known L-to-D-amino-acid-residue isomerase in a mammal. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3

Relaxin- 3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin- 1 and - 2, whose main functions are associated with pregnancy, relaxin- 3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin- 3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin- 3 adopts an insulin- like fold, but the structure differs crucially from the crystal structure of human relaxin- 2 near the B- chain terminus. In particular, the B- chain C terminus folds back, allowing Trp(B27) to interact with the hydrophobic-core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin- 3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135.

The importance of host ecology in thelastomatoid (Nematoda : Oxyurida) host specificity

An experimental investigation of host specificity within the Thelastomatoidea is presented by means of a comparison of the thelastomatoids of two panesthiine cockroaches, Panesthia cribrata and R tryoni tryoni, with those of other log-dwelling arthropods and those of leaf litter dwelling arthropods found near by. 145 log-dwelling and leaf-litter dwelling arthropods, representing adjacent ecological niches, were collected from Lamington National Park, Queensland, Australia. A high degree of thelastomatoid species sharing (19 incidences from 26 specimens) occurs between log-dwelling arthropods and the two cockroach species. No overlap in thelastomatoid fauna was observed between the log dwelling and leaf-litter dwelling groups. Our results suggest that host specificity of thelastomatoids is largely dictated by host ecology. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Venom proteins from polydnavirus-producing endoparasitoids: Their role in host-parasite interactions

Endoporasitoid wasps have evolved various mechanisms to ensure successful development of their progeny, including co-injection of a cocktail of maternal secretions into the host hemocoel, including venom, calyx fluid, and polydnoviruses. The components of each type of secretion may influence host physiology and development independently or in a synergistic fashion. For example, venom fluid consists of several peptides and proteins that promote expression of polydnavirus genes in addition to other activities, such as inhibition of prophenoloxidase activation, inhibition of hemocytes spreading and aggregation, and inhibition of development. This review provides a brief overview of advances and prospects in the study of venom proteins from polydnavirus-producing endoparositoid wasps with a special emphasis on the role of C. rubecula venom proteins in host-parositoid interactions.

A calreticulin-like protein from endoparasitoid venom fluid is involved in host hemocyte inactivation

During oviposition, most endoparasitoid wasps inject maternal factors into their hosts to interfere with host immune reactions and ensure successful development of their progeny. Since encapsulation is a major cellular defensive response of insects against intruding parasites, parasitoids have developed numerous mechanisms to suppress the host encapsulation capability by interfering with every step in the process, including recognition, adherence and spreading. In previous studies, components of Cotesia rubecula venom were shown to inhibit melanization of host hemolymph by interfering with the prophenoloxidase activation cascade and facilitate expression of polydnavirus genes. Here we report the isolation and characterization of another venom protein with similarity to calreticulin. Results indicate that C rubecula calreticulin (CrCRT) inhibits hemocyte spreading behavior, thus preventing encapsulation of the developing parasitoid. It is possible that the protein might function as an antagonist competing for binding sites with the host hemocyte calreticulin, which mediates early-encapsulation reactions. (c) 2005 Elsevier Ltd. All rights reserved.

Comparison of active venom components between Eastern brown snakes collected from South Australia and Queensland

The abundance and activity of the prothrombin activator (pseutarin C) within the venom of the Eastern brown snake (Pseudonaja textilis textilis) is the primary determinant of its coagulation potency. Textilinin-1, also in this venom, is a plasmin inhibitor which is thought to exert its toxic effects through the slowing of fibrinolysis. The aim of this report is to determine if there are differences in the potency of the venom from Eastern brown snakes collected from South Australia (SA) compared to those from Queensland (QLD). A concentration of 0.4 mu g/ml venom protein from six QLD specimens clotted citrated plasma in an average time of 21.4 +/- 3.3 s compared to 68.7 +/- 2.4 s for the same amount of SA venom (averaged for six individuals). The more potent procoagulant activity of the QLD venom was measured between 0.4 and 94 mu g/ml venom protein in plasma. The anti-plasmin activity of textilinin was also greater in the venom of the snakes collected from QLD, causing full inhibition of plasmin at approximately 1.88 mu g/ml of venom protein compared to approximately 7.5 mu g/ml for the SA venoms. It is concluded that geographic differentiation of the Eastern brown snakes results in significant differences venom potency.