Venom of the Red-Bellied Black Snake Pseudechis porphyriacus Shows Immunosuppressive Potential.

Venoms act with remarkable specificity upon a broad diversity of physiological targets. Venoms are composed of proteins, peptides, and small molecules, providing the foundation for the development of novel therapeutics. This study assessed the effect of venom from the red-bellied black snake (Pseudechis porphyriacus) on human primary leukocytes using bead-based flow cytometry, mixed lymphocyte reaction, and cell viability assays. We show that venom treatment had a significant immunosuppressive effect, inhibiting the secretion of interleukin (IL)-2 and tumor necrosis factor (TNF) from purified human T cells by 90% or greater following stimulation with mitogen (phorbol 12-myristate 13-acetate and ionomycin) or via cluster of differentiation (CD) receptors, CD3/CD28. In contrast, venom treatment did not inhibit TNF or IL-6 release from antigen-presenting cells stimulated with lipopolysaccharide. The reduced cytokine release from T cells was not associated with inhibition of T cell proliferation or reduction of cell viability, consistent with an anti-inflammatory mechanism unrelated to the cell cycle. Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity. These data suggest that compounds from P. porphyriacus venom may be potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease.

A randomised, placebo-controlled trial assessing the efficacy of an oral B group vitamin in preventing the development of chemotherapy-induced peripheral neuropathy (CIPN).

INTRODUCTION: Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect resulting from neurotoxic chemotherapeutic agents. This study aimed to assess the efficacy and safety of an oral B group vitamin compared to placebo, in preventing the incidence of CIPN in cancer patients undergoing neurotoxic chemotherapy. METHODS: A pilot, randomised, placebo-controlled trial was conducted. Newly diagnosed cancer patients prescribed with taxanes, oxaliplatin or vincristine were invited to participate. A total of 71 participants (female 68 %, male 32 %) were enrolled into the study and randomised to the B group vitamin (n = 38) arm or placebo (n = 33). The data from 47 participants were eligible for analysis (B group vitamins n = 27, placebo n = 22). The primary outcome measure was the total neuropathy score assessed by an independent neurologist. Secondary outcome measures included serum vitamin B levels, quality of life, pain inventory and the patient neurotoxicity questionnaires. Outcome measures were conducted at baseline, 12, 24 and 36 weeks. RESULTS: The total neuropathy score (TNS) demonstrated that a B group vitamin did not significantly reduce the incidence of CIPN compared to placebo (p = 0.73). Statistical significance was achieved for patient perceived sensory peripheral neuropathy (12 weeks p = 0.03; 24 weeks p = 0.005; 36 weeks p = 0.021). The risk estimate for the Patient Neurotoxicity Questionnaire (PNQ) was also statistically significant (OR = 5.78, 95 % CI = 1.63-20.5). The European Organisation of Research and Treatment of Cancer (EORTC) quality of life, total pain score and pain interference showed no significance (p = 0.46, p = 0.9, p = 0.37 respectively). A trend was observed indicating that vitamin B12 may reduce the onset and severity of CIPN. CONCLUSION: An oral B group vitamin as an adjunct to neurotoxic chemotherapy regimens was not superior to placebo (p > 0.05) for the prevention of CIPN. Patients taking the B group vitamin perceived a reduction in sensory peripheral neuropathy in the PNQ. Moreover, a robust clinical study is warranted given that vitamin B12 may show potential in reducing the onset and severity of CIPN. Trial number: ACTRN12611000078954 Protocol number: UH2010000749.

Current and potential uses of bioactive molecules from marine processing waste.

Food industries produce huge amounts of processing waste that are often disposed of incurring expenses and impacting upon the environment. For these and other reasons, food processing waste streams, in particular marine processing waste streams, are gaining popularity amongst pharmaceutical, cosmetic and nutraceutical industries as sources of bioactive molecules. In the last 30 years, there has been a gradual increase in processed marine products with a concomitant increase in waste streams that include viscera, heads, skins, fins, bones, trimmings and shellfish waste. In 2010, these waste streams equated to approximately 24 million tonnes of mostly unused resources. Marine processing waste streams not only represent an abundant resource, they are also enriched with structurally diverse molecules that possess a broad panel of bioactivities including anti-oxidant, anti-coagulant, anti-thrombotic, anti-cancer and immune-stimulatory activities. Retrieval and characterisation of bioactive molecules from marine processing waste also contributes valuable information to the vast field of marine natural product discovery. This review summarises the current use of bioactive molecules from marine processing waste in different products and industries. Moreover, this review summarises new research into processing waste streams and the potential for adoption by industries in the creation of new products containing marine processing waste bioactives.

TGFß isoforms and receptors mRNA expression in breast tumours: prognostic value and clinical implications.

BACKGROUND: Transforming growth factor beta (TGFß) signalling is involved in both tumour suppression and tumour progression. The mRNA expression levels of the TGFß isoforms and receptors in breast tumours may have prognostic value and clinical implications. METHODS: The mRNA levels of TGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 were analysed in primary breast tumours and adjacent normal breast tissues, and the associations with tumour characteristics and patients' overall and relapse-free survival were evaluated, using the public gene expression microarray data from The Cancer Genome Atlas (n = 520) and the Gene Expression Omnibus (four datasets) and our quantitative real-time PCR validation data (n = 71). RESULTS: Significantly higher TGFB1 and TGFB3 mRNA levels and lower TGFBR2 mRNA levels were observed in primary tumours compared with their paired normal tissues. TGFB1 mRNA expression was seemly lower in triple-negative tumours and in tumours from lymph node-negative patients. TGFB3 mRNA expression was significantly lower in estrogen receptor-negative/progesterone receptor-negative/Basal-like/Grade 3 tumours. High TGFB2, TGFB3 and TGFBR2 mRNA levels in tumours were generally associated with better prognosis for patients, especially those diagnosed with lymph node-negative diseases. High TGFBR1 mRNA levels in tumours were associated with poorer clinical outcomes for patients diagnosed with small (diameter ≤ 2 cm) tumours. CONCLUSIONS: The results indicate a reduced responsiveness of tumour cells to TGFß, a preferential up-regulation of TGFB1 in malignant tumours and a preferential up-regulation of TGFB3 in premalignant tumours. The results may not only provide prognostic value for patients but also assist in classifying tumours according to their potential responses to TGFß and selecting patients for TGFß signalling pathway targeted therapies.

Marine-Based Nutraceuticals: An Innovative Trend in the Food and Supplement Industries.

Recent trends in functional foods and supplements have demonstrated that bioactive molecules play a major therapeutic role in human disease. Nutritionists and biomedical and food scientists are working together to discover new bioactive molecules that have increased potency and therapeutic benefits. Marine life constitutes almost 80% of the world biota with thousands of bioactive compounds and secondary metabolites derived from marine invertebrates such as tunicates, sponges, molluscs, bryozoans, sea slugs and many other marine organisms. These bioactive molecules and secondary metabolites possess antibiotic, antiparasitic, antiviral, anti-inflammatory, antifibrotic and anticancer activities. They are also inhibitors or activators of critical enzymes and transcription factors, competitors of transporters and sequestrants that modulate various physiological pathways. The current review summaries the widely available marine-based nutraceuticals and recent research carried out for the purposes of isolation, identification and characterization of marine-derived bioactive compounds with various therapeutic potentials.

ß-Adducin siRNA disruption of the spectrin-based cytoskeleton in differentiating keratinocytes prevented by calcium acting through calmodulin/epidermal growth factor receptor/cadherin pathway.

Here, we report that siRNA transfection of ß-adducin significantly disrupted the spectrin-based cytoskeleton and cytoskeletal arrangements of both ß-adducin and PKCδ by substantially inhibiting the expression of ß-adducin, spectrin and PKCδ proteins in differentiating keratinocytes. However, extracellular Ca2+ treatment blocked the inhibitory effects of the ß-adducin siRNA. Ca2+ also prevented the significant down-regulation of two differentiation markers involucrin and K1/10 and the distinct up-regulation of proliferation marker K14 in ß-adducin siRNA transfected keratinocytes. In addition, ß-adducin knockdown resulted in a substantial reduction of epidermal growth factor receptor (EGFR), cadherin and ß-catenin and enhanced phosphorylation of EGFR on tyrosine 1173 and Ca2+ prevented these changes. Furthermore, Ca2+ blocked the inhibitory effects of ß-adducin siRNA on the expression of calmodulin, phosphorylated-calmodulin (P-CaM((Tyr138))) and myristoylated alanine-rich C-kinase substrate (MARCKS) in keratinocytes. Co-immunoprecipitation studies further revealed that calmodulin, not MARCKS, strongly interacted with EGFR, cadherin and ß-catenin. Our data suggest that Ca2+ plays an important role in regulating the expression and function of ß-adducin to sustain normal organization of the spectrin-based cytoskeleton and the differentiation properties in keratinocytes through the calmodulin/EGFR/cadherin signaling pathway.

Four major factors regulate phosphatidylinositol 3-kinase signaling pathway in cancers induced by infection of human papillomaviruses.

Epidemiological surveys and molecular studies have indicated that infection of human papillomavirus (HPV)itself is necessary but insufficient for completing transformation of the human epithelial cells in vivo to lead to different cancers. Mounting evidence exists that HPV E6/E7 oncoproteins indeed alter the cellular and molecular events in their transformed cells to induce cancers through a phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. The PI3K/AKT/mTOR signaling pathway is, nonetheless, of the central importance, which tightly modulates many cellular events that occur in cells to lead them to be cancerous under the action of oncogenic factors. The cancinogenic roles of the PI3K/AKT/mTOR signaling in HPV-induced cancers are generally regulated by different upstream signaling molecules such as upstream receptor tyrosine kinases. In this article, we review that the four major upstream signaling molecules (growth factor receptor, notch receptor, Ras and PI3KCA genes) regulate PI3K/AKT/mTOR pathway to confer oncogenicity in HPV-immortalized epithelial cells and various transformed phenotypes.

Calcium prevents retinoic acid-induced disruption of the spectrin-based cytoskeleton in keratinocytes through the Src/PI3K-p85α/AKT/PKCδ/ß-adducin pathways.

We have previously reported that spectrin increases dramatically in amount and is assembled into the cytoskeleton in differentiating keratinocytes both in vitro and in vivo (Zhao et al., PLoS ONE 6 (12) (2011) e28267). We demonstrate here that extracellular calcium (Ca2+) enhances differentiation of keratinocytes and that this is associated with increased spectrin expression and formation of a spectrin-based cytoskeleton. While Retinoic acid (RA) also enhanced keratinocyte differentiation, it abrogated the spectrin-based cytoskeleton in keratinocytes. Furthermore, RA substantially inhibited expression of both Src and PI3K-p85α and consequently the amounts of specific phosphorylation of both of these proteins. RA also enhanced AKT expression and dramatically induced phosphorylation of AKT((Thr308)), accompanied by phosphorylation of both PKCδ((Thr505)) and ß-adducin((Ser662)) and upregulated cyclin D2 and down-regulated cyclin B1. On the other hand, Ca2+ overcame the inhibitory effects of RA on expression of Src, PI3K-p85α and cyclin B1 by maintaining high levels of phosphorylation of both Src((Tyr527)) and PI3K-p85α and preventing phosphorylation of AKT((Thr308)), PKCδ((Thr505)) and ß-adducin((Ser662)). These data highlight the importance of Ca2+ in both spectrin expression and the organizational integrity of the spectrin-based cytoskeleton in differentiating keratinocytes and assist in elucidating the signalling pathways involved.

Nutraceuticals and chemotherapy induced peripheral neuropathy (CIPN): a systematic review.

Chemotherapy induced peripheral neuropathy [CIPN] is a common significant and debilitating side effect resulting from the administration of neurotoxic chemotherapeutic agents. These pharmaco-chemotherapeutics can include taxanes, vinca alkaloids and others. Moderate to severe CIPN significantly decreases the quality of life and physical abilities of cancer patients and current pharmacotherapy for CIPN e.g. Amifostine and antidepressants have had limited efficacy and may themselves induce adverse side effects. To determine the potential use of nutraceuticals i.e. vitamin E, acetyl-L-carnitine, glutamine, glutathione, vitamin B6, omega-3 fatty acids, magnesium, calcium, alpha lipoic acid and n-acetyl cysteine as adjuvants in cancer treatments a systematic literature review was conducted. Revised clinical studies comprised of randomized clinical trials that investigated the anti-CIPN effect of nutraceuticals as the adjuvant intervention in patients administered chemotherapy. Twenty-four studies were assessed on methodological quality and limitations identified. Studies were mixed in their recommendations for nutraceuticals. Currently no agent has shown solid beneficial evidence to be recommended for the treatment or prophylaxis of CIPN. The standard of care for CIPN includes dose reduction and/or discontinuation of chemotherapy treatment. The management of CIPN remains an important challenge and future studies are warranted before recommendations for the use of supplements can be made.

Drug development from Australian elapid snake venoms and the Venomics pipeline of candidates for haemostasis: Textilinin-1 (Q8008), Haempatch™ (Q8009) and CoVase™ (V0801).

Snake venoms are attractive for drug discovery and development, with a number of therapeutics derived from snake venom either in clinical use or in development. Recognising this opportunity, Australian biopharmaceutical company QRxPharma Ltd and its subsidiary Venomics Pty Ltd (VPL) has partnered with the University of Queensland (UQ) to screen and develop drug candidates from Australian elapid snake venoms. VPL has three haemostasis candidates in early preclinical development. Textilinin-1 (Q8008) is a 7 kDa potent and selective plasmin inhibitor that has application as an anti-fibrinolytic agent to reduce blood loss associated with complex surgeries. Haempatch™ (Q8009) is a Factor Xa-like protein that displays potent procoagulant effects and is being developed as a topical haemostatic agent to reduce blood loss resulting from surgery or trauma. CoVase™ (V0801) is a procoagulant cofactor that may have application as a systemic anti-bleeding agent in the treatment of internal bleeding and non-compressible haemorrhage. This review focuses on drug discovery from Australian elapid snake venoms, with emphasis on the QRxPharma/VPL drug discovery project undertaken in collaboration with UQ and candidates at further stages of development.

Cloning and characterisation of novel cystatins from elapid snake venom glands.

Snake venoms contain a complex mixture of polypeptides that modulate prey homeostatic mechanisms through highly specific and targeted interactions. In this study we have identified and characterised cystatin-like cysteine-protease inhibitors from elapid snake venoms for the first time. Novel cystatin sequences were cloned from 12 of 13 elapid snake venom glands and the protein was detected, albeit at very low levels, in a total of 22 venoms. One highly conserved isoform, which displayed close sequence identity with family 2 cystatins, was detected in each elapid snake. Crude Austrelaps superbus (Australian lowland copperhead) snake venom inhibited papain, and a recombinant form of A. superbus cystatin inhibited cathepsin L â‰… papain > cathepsin B, with no inhibition observed for calpain or legumain. While snake venom cystatins have truncated N-termini, sequence alignment and structural modelling suggested that the evolutionarily conserved Gly-11 of family 2 cystatins, essential for cysteine protease inhibition, is conserved in snake venom cystatins as Gly-3. This was confirmed by mutagenesis at the Gly-3 site, which increased the dissociation constant for papain by 10(4)-fold. These data demonstrate that elapid snake venom cystatins are novel members of the type 2 family. The widespread, low level expression of type 2 cystatins in snake venom, as well as the presence of only one highly conserved isoform in each species, imply essential housekeeping or regulatory roles for these proteins.

Evaluation of the Becton-Dickinson rapid serum tube: does it provide a suitable alternative to lithium heparin plasma tubes?

BACKGROUND: Obtaining a suitable specimen for analysis in a timely manner is pivotal in clinical chemistry service provision. Serum is recognized as the preferred specimen for most assays, but because of time constraints for completion of clotting and an increasing number of patients on anti-coagulant therapy, latent clotting or no clotting is an outcome which can lead to errors and delay in delivery of critical results. Although lithium heparin plasma has unique problems, it has become an alternative in hospital-based laboratories. METHODS: The Becton-Dickinson (BD) rapid serum tube (RST) was evaluated in a hospital environment using a total of 53 participants, both healthy and anticoagulated, for 31 analytes against BD PST II and BD SST II tubes measured with Beckman DxC800 and DxI800 analyzers. RESULTS: Most results from the RST tube were comparable with those from the SST II tube. Potassium results were closer to the PST II plasma concentrations. Incomplete and latent clotting was encountered in the RST specimens from participants (cardiac and dialysis) who had received a total of >7000 units of heparin [activated partial thromboplastin time (APTT) >150 s], warfarin/heparin combination, and specimens from cardiac surgery patients who had received a total of >25,000 units of heparin (APTT >200 s) at the time of collection of specimens. CONCLUSIONS: The RST tube provides a suitable alternative to lithium heparin plasma tubes for most patients in a hospital environment. However, latent clotting continued to occur in specimens collected from participants who had received high concentrations of anticoagulants.

Altered clot kinetics in patients with non-alcoholic fatty liver disease.

BACKGROUND: Emerging evidence has linked the presence of non-alcoholic fatty liver disease (NAFLD) with an increased risk for cardiovascular events. We hypothesised that altered clot kinetics and platelet function may contribute to this increased risk. This study compared whole blood clotting kinetics in patients with 1) non-cirrhotic NAFLD (n = 28) and 2) healthy control subjects (n = 22). METHODS: Clotting kinetics were assessed in whole blood using thromboelastography (TEG) and assessed for correlations with cardiovascular risk factors. RESULTS: Clot kinetics in patients with NAFLD showed significantly stronger clot development (maximum amplitude (MA); 58.3 +/- 6.3 mm vs. 52.0 +/- 10.1 mm, p = 0.01) and reduced clot lysis in the presence of thrombin (35 +/- 30% vs. 51 +/- 26% clot lysis 30 minutes after MA, p = 0.03) compared to control subjects. Clot strength was independently positively associated with body mass index in NAFLD, but not in control subjects. There was a greater platelet contribution to clot strength in patients with NAFLD compared to controls despite similar platelet counts. There was no association between clot kinetics and features of the metabolic syndrome or presence of type 2 diabetes. CONCLUSION: Patients with NAFLD have disturbances in ex-vivo clot kinetics including increased clot strength and clots that are more resistant to thrombin-stimulated lysis.

Crystal structure of textilinin-1, a Kunitz-type serine protease inhibitor from the venom of the Australian common brown snake (Pseudonaja textilis).

Textilinin-1 is a Kunitz-type serine protease inhibitor isolated from the venom of the Australian common brown snake, Pseudonaja textilis. This molecule binds to and blocks the activity of a range of serine proteases, including plasmin and trypsin. Textilinin-1's ability to inhibit plasmin, a protease involved in fibrinolysis, has raised the possibility that it could be used as an alternative to aprotinin (Trasylol) as a systemic antibleeding agent in surgery. Here, the crystal structure of free recombinant textilinin-1 has been determined to 1.63 A, with three molecules observed in the asymmetric unit. All of these have a similar overall fold to aprotinin, except that the canonical loop for one of the molecules is inverted such that the side chain of the P1' residue, Val18, is partially buried by intramolecular contacts to Pro15, Thr13, and Ile36. In aprotinin, the P1' residue is Ala16, whose side chain is too small to form similar contacts. The loop inversion in textilinin-1 is facilitated by changes in backbone dihedral angles for the P1 and P2' residues, such that they alternate between values in the beta-sheet and alpha-helical regions of the Ramachandran plot. In a comparison with the structures of all other known Kunitz-type serine protease inhibitors, no such conformational variability has been observed. The presence of the bulkier valine as the P1' residue in textilinin-1 appears to be a major contributor to reducing the binding affinity for plasmin as compared to aprotinin (3.5 nm versus 0.053 nm) and could also account for an observed narrower binding specificity.

Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss.

Aprotinin has been used widely in surgery as an anti-bleeding agent but is associated with a number of side effects. We report that textilinin-1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin-1 at 5 micromol/l gave almost complete inhibition of tissue plasminogen activator-induced fibrinolysis of whole blood clots. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin-1. In a mouse tail-vein bleeding model, intravenous textilinin-1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin-treated animals was significantly shorter than in aprotinin-treated mice. Based on these data, textilinin-1 merits further investigation as a therapeutic alternative to aprotinin.

Diversity of toxic components from the venom of the evolutionarily distinct black whip snake, Demansia vestigiata.

Included among the more than 300 species of elapid snakes worldwide is the Australian genus Demansia, or whip snakes. Despite evidence to suggest adverse clinical outcomes from envenomation by these snakes, together with confusion on their true phylogenetic relationship to other Australian elapids, not a single toxin sequence has previously been reported from the venom of a Demansia species. We describe here a combined proteomic and transcriptomic approach characterizing the venom from the black whip snake, Demansia vestigiata. A total of 13 distinct toxin families were identified, including homologues of all of the major toxic components previously reported from the venom of other Australian elapids, such as factor X-like prothrombin activators, neurotoxins, phospholipases, cysteine rich secretory proteins, textilinin-like molecules, nerve growth factors, l-amino acid oxidases, vespryns, 5' nucleotidases, metalloproteinases, and C-type lectins as well as a novel dipeptidyl peptidase family. Phylogenetic analysis of these sequences revealed an early evolutionary split of the black whip snake from all other characterized Australian snakes, with a low degree of sequence identity between D. vestigiata and the other snakes, across all toxin families. The results of this study have important implications not only for the further characterization of venom from whip snakes, but also for our understanding of the evolutionary relationship of Australian snake species.

The diversity of bioactive proteins in Australian snake venoms.

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.

Surfactant protein expression in human skin: evidence and implications.

The presence of surfactant proteins (SPs), critical to local barrier and defense functions and usually associated with the lung, was revealed in adult and fetal human skin complementary deoxyribonucleic acid, in skin samples from three adult female donors and also in cultured fibroblasts, keratinocytes, and melanocytes. Using reverse transcription-PCR, SP-A, SP-B, SP-C, and SP-D messenger ribonucleic acid expression was detected to varying extents in the different skin sources. The stronger expression of SP-C in fetal skin, compared to adult skin, suggested that the role of this protein alters with age. Immunohistochemical studies showed variable distribution of SPs in human epidermis and dermis, confirming that these proteins are indeed translated and expressed in skin tissue. In vitro studies showed that the surface tension of SP-deficient artificial sebum is (a) lowered by skin-extracted SP-B and (b) further reduced to a level comparable to normal sebum by the additional presence of skin-extracted SP-A and SP-D, consistent with their surface tension-lowering capabilities in lung. The possible roles of SPs in skin, based on their known functions in the lung are discussed. However, their potential as therapeutic targets or diagnostic markers of skin disease remains to be elucidated.

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 N-linked glycosylation. It is possible that these modifications alter the stability, activity and other characteristics of the snake NGFs. Further characterisation is necessary to delineate the function of the individual NGF isoforms.

Cloning and characterisation 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.