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.

Isolation and characterization at cholinergic nicotinic receptors of a neurotoxin from the venom of the Acanthophis sp Seram death adder

The present study describes the isolation of the first neurotoxin (acantoxin IVa) from Acanthophis sp. Seram death adder venom and an examination of its activity at nicotinic acetylcholine receptor (naChR) subtypes. Acantoxin IVa (MW 6815; 0.1-1.0 muM) caused concentration-dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms, supramaximal V) and inhibited contractile responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle, confirming that this toxin is a postsynaptic neurotoxin. Acantoxin IVa (1-10 nM) caused pseudo-irreversible antagonism at skeletal muscle nAChR with an estimated pA(2) Of 8.36 +/- 0.17. Acantoxin IVa was approximately two-fold less potent than the long-chain (Type 11) neurotoxin, alpha-bungarotoxin. With a pK(i) value of 4.48, acantoxin IVa was approximately 25,000 times less potent than a-bungarotoxin at alpha7-type neuronal nAChR. However, in contrast to alpha-bungarotoxin, acantoxin IVa completely inhibited specific [H-3]-methyllycaconitine (MLA) binding in rat hippocampus homogenate. Acantoxin IVa had no activity at ganglionic nAChR, alpha4beta2 subtype neuronal nAChR or cytisine-resistant [H-3]-epibatidine binding sites. While long-chain neurotoxin resistant [H-3]-MLA binding in hippocampus homogenate requires further investigation, we have shown that a short-chain (Type 1) neurotoxin is capable of fully inhibiting specific [H-3]-MLA binding. (C) 2004 Elsevier Inc. All rights reserved.

Therapeutic effects and possible mechanisms of a snake venom preparation in the fibrotic rat liver

The effects of a Chinese snake venom preparation from Agkistrodon halys pallas, used for treatment of hepatic fibrosis/cirrhosis in China, was investigated in an {in vivo} rat model and using in situ hepatic perfusion. Four groups were used in the experiments: (i) healthy, (ii) healthy/venom-treated, (iii) carbon tetrachloride (CCl4)-treated, and (iv) CCl4/venom-treated. Treatment effects were assessed by determining hepatic histopathology, biochemistry and fibrosis index parameters, bile production, biliary taurocholate recovery, hepatic mRNA expression of four bile salt transporters (Ntcp, Bsep, Oatp-1, and Oatp-3), comparison of hepatic microcirculation, fibrinolytic activity, and antithrombotic effects. Liver histopathology, biochemistry, and fibrosis index showed a dramatic improvement in venom-treated animals. There were significant differences in bile production between healthy/venom-treated and all other experimental groups and between CCl4/venom-treated and CCl4-treated animals, but no significant differences were found between CCl4/venom-treated and healthy animals. Biliary taurocholate recovery was significantly increased in healthy/venom-treated and CCl4/venom-treated animals. The expression of mRNA levels of the four bile salt transporters showed an increase after venom treatment. The hepatic microcirculation studies showed normalized sinusoidal beds in CCl4/venom-treated animals compared to healthy animals, whereas CCl4-treated animals showed abnormal profiles to the healthy and the CCl4/AHPV-treated animals. The fibrinogen and plasma thromboxane B-2 levels of healthy rats decreased with increasing dose after venom treatment. It was concluded that snake venom treatment may be therapeutic in treatment of hepatic fibrosis/cirrhosis by possibly a combination of increased bile flow and improved hepatic microcirculation, changes in bile salt transporter expression, and fibrinolytic and antithrombotic effects of the snake venom preparation.

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.

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.

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.

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.

Molecular size as the main determinant of solute maximum flux across the skin

One of the most important determinants of dermatological and systemic penetration after topical application is the delivery or flux of solutes into or through the skin. The maximum dose of solute able to be delivered over a given period of time and area of application is defined by its maximum flux (J(max), mol per cm(2) per h) from a given vehicle. In this work, J(max) values from aqueous solution across human skin were acquired or estimated from experimental data and correlated with solute physicochemical properties. Whereas epidermal permeability coefficients (k(p)) are optimally correlated to solute octanol-water partition coefficient (K-ow) and molecular weight (MW) was found to be the dominant determinant of J(max) for this literature data set: log J(max)=-3.90-0.0190MW (n=87, r(2)=0.847, p

Snakes and cats in the flower bed: Fast detection is not specific to pictures of fear-relevant animals

The observation that snakes and spiders are found faster among flowers and mushrooms than vice versa and that this search advantage is independent of set size supports the notion that fear-relevant stimuli are processed preferentially in a dedicated fear module. Experiment I replicated the faster identification of snakes and spiders but also found a set size effect in a blocked, but not in a mixed-trial, sequence. Experiment 2 failed to find faster identification of snake and spider deviants relative to other animals among flowers and mushrooms and provided evidence for a search advantage for pictures of animals, irrespective of their fear relevance. These findings suggest that results from the present visual search task cannot support the notion of preferential processing of fear relevance.

Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.

Physiological evidence for reproductive suppression in the introduced population of brown tree snakes (Boiga irregularis) on Guam

Introduced species are an increasingly pervasive problem. While studies on the ecology and behavior of these pests are numerous, there is relatively little known of their physiology, specifically their reproductive and stress physiology. One of the best documented introduced pest species is the brown tree snake, Boiga irregularis, which was introduced onto the Pacific island of Guam sometime around World War II. The snake is responsible for severely reducing Guam's native vertebrates. We captured free-living individuals throughout the year and measured plasma levels of stress and sex hormones in an effort to determine when they were breeding. These data were compared to reproductive cycles from a captive population originally collected from Guam. Free-living individuals had chronically elevated plasma levels of the stress hormone corticosterone and basal levels of sex steroids and a remarkably low proportion were reproductively active. These data coincide with evidence that the wild population may be in decline. Captive snakes, had low plasma levels of corticosterone with males displaying a peak in plasma testosterone levels during breeding. Furthermore, we compared body condition between the free-living and captive snakes from Guam and free-living individuals captured from their native range in Australia. Male and female free-living snakes from Guam exhibited significantly reduced body condition compared to free-living individuals from Australia. We suggest that during the study period, free-living brown tree snakes on Guam were living under stressful conditions, possibly due to overcrowding and overexploitation. of food resources, resulting in decreased body condition and suppressed reproduction. (C) 2004 Elsevier Ltd. All rights reserved.