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.

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.

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.

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.

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.

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.

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.

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.

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.

Negative regulation of prophenoloxidase (proPO) activation by a clip-domain serine proteinase homolog (SPH) from endoparasitoid venom

Most parasitic wasps inject maternal factors into the host hemocoel to suppress the host immune system and ensure successful development of their progeny. Melanization is one of the insect defence mechanisms against intruding pathogens or parasites. We previously isolated from the venom of Cotesia rubecula a 50 kDa protein that blocked melanization in the hemolymph of its host, Pieris rapae [Insect Biochem. Mol. Biol. 33 (2003) 1017]. This protein, designated Vn50, is a serine proteinase homolog (SPH) containing an amino-terminal clip domain. In this work, we demonstrated that recombinant Vn50 bound P. rapae hemolymph components that were recognized by antisera to Tenebrio molitor prophenoloxidase (proPO) and Manduca sexta proPO-activating proteinase (PAP). Vn50 is stable in the host hemolymph-it remained intact for at least 72 It after parasitization. Using M. sexta as a model system, we found that Vn50 efficiently down-regulated proPO activation mediated by M. sexta PAP-1, SPH-1, and SPH-2. Vn50 did not inhibit active phenoloxidase (PO) or PAP-1, but it significantly reduced the proteolysis of proPO. If recombinant Vn50 binds P. rapae proPO and PAP (as suggested by the antibody reactions), it is likely that the molecular interactions among M. sexta proPO, PAP-1, and SPHs were impaired by this venom protein. A similar strategy might be employed by C rubecula to negatively impact the proPO activation reaction in its natural host. (C) 2004 Elsevier Ltd. All rights reserved.

A novel venom peptide from an endoparasitoid wasp is required for expression of polydnavirus genes in host hemocytes

Maternal factors introduced into host insects by endoparasitoid wasps are usually essential for successful parasitism. This includes polydnaviruses (PDVs) that are produced in the reproductive organ of female hymenopteran endoparasitoids and are injected, together with venom proteins, into the host hemocoel at oviposition. Inside the host, PDVs enter various tissue cells and hemocytes where viral genes are expressed, leading to developmental and physiological alterations in the host, including the suppression of the host immune system. Although several studies have shown that some PDVs are only effective when accompanied by venom proteins, there is no report of an active venom ingredient(s) facilitating PDV infection and/or gene expression. In this study, we describe a novel peptide (Vn1.5) isolated from Cotesia rubecula venom that is required for the expression of C. rubecula bracoviruses (CrBVs) in host hemocytes (Pieris rapae), although it is not essential for CrBV entry into host cells. The peptide consists of 14 amino acids with a molecular mass of 1598 Da. In the absence of Vn1.5 or total venom proteins, CrBV genes are not expressed in host cells and did not cause inactivation of host hemocytes.

Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase

Reasons for performing study: The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. Objectives: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. Methods: Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression. Results: Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P