Crystallization and preliminary X-ray diffraction analysis of a myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom.
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Crystals of a myotoxic phospholipase A(2) from Bothrops neuwiedi pauloensis have been obtained. They diffracted at 2.5 A resolution using a synchrotron radiation source and belong to space group P3(1)21. Preliminary analysis shows that there are two molecules in the asymmetric unit. (+info)
Tyr-->Trp-substituted peptide 115-129 of a Lys49 phospholipase A(2) expresses enhanced membrane-damaging activities and reproduces its in vivo myotoxic effect.
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Myotoxin II is a group II Lys49 phospholipase A(2) (PLA(2)) isolated from the venom of the snake Bothrops asper. Previous studies on a synthetic peptide derived from its heparin-binding, cationic/hydrophobic sequence 115-129 demonstrated a direct functional role of this particular region in the in vitro cytolytic and bactericidal actions of the protein. Nevertheless, no significant myonecrosis has been observed after local intramuscular injection of peptide 115-129 (p115-129) in mice. Since the membrane-damaging action of p115-129 was proposed to depend on its amphiphilic character, the present study examined the effects of substituting its cluster of three tyrosine residues by tryptophan residues, on its toxic/pharmacological activities in vitro and in vivo. This substitution resulted in a drastic enhancement of the membrane-damaging activities of the peptide (p115-W3), together with the clear expression of myotoxic activity in vivo. Both the heparin-binding and antigenic characteristics of p115-129 were essentially conserved in p115-W3, suggesting that the modification did not lead to radical structural alterations. In addition to myotoxicity, cytotoxicity, and bactericidal action, p115-W3 exerted edema-forming activity in the mouse footpad assay. Thus, the synthetic 13-mer p115-W3 reproduced all the known toxic effects of myotoxin II. In spite of its potent membrane-damaging actions, p115-W3 did not acquire direct hemolytic activity upon mouse erythrocytes, an effect which is not present in myotoxin II, but that has been ascribed to the presence of tryptophan in other cationic, membrane-damaging peptides such as mellitin from bee venom. The myotoxic activity of p115-W3 herein described constitutes the first example of a short, PLA(2)-based linear synthetic peptide with the ability to reproduce this effect of a parent protein in vivo. This finding is in clear support of the proposed relevance of the C-terminal region 115-129 in all the membrane-damaging mechanisms exerted by myotoxin II, including the myotoxic mechanism. (+info)
Monoclonal antibodies against the Androctonus australis hector scorpion neurotoxin I: characterisation and use for venom neutralisation.
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A series of monoclonal antibodies (mAbs) specific for the alpha-neurotoxin I (Aah I) from the venom of the dangerous Androctonus australis hector scorpion were obtained using carrier protein-coupled toxin. Competitive RIA, receptor assays and mouse toxicity tests were performed to characterise mAbs in terms of affinity and neutralisation. Cross-reactivity studies and two-site ELISA results allowed some classification of mAbs into three groups. One mAb, 9C2, was particularly interesting since it recognised the parent toxin I with a K(D) of 0.15 nM and was also reactive with toxins of the same immunological group. Its ability to neutralise the toxic effect of the parent toxin and the venom fraction has been investigated. This anti-Aah I mAb 9C2, associated with anti-Aah II mAb 4C1, provides a valuable tool to neutralise the toxicity of the venom. (+info)
Xenopus Xenf: an early endodermal nuclear factor that is regulated in a pathway distinct from Sox17 and Mix-related gene pathways.
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We report a novel zygotic gene encoding a Xenopus endodermal nuclear factor, Xenf. Expression of Xenf starts at the late blastula stages and is decreased after gastrulation. Xenf shows no structural homology to any known proteins. When GFP-tagged Xenf is overexpressed in Xenopus cells, Xenf protein is localized to the nucleus, associating closely with the chromosomes. In animal cap assays, Xenf expression is strongly activated by mRNA injection of Vg1 and VegT, maternal vegetal genes that can induce endodermal differentiation. In contrast, Xenf is not induced by endoderm-inducing zygotic transcription factors such as Sox17 and Mix-related genes. In turn, Xenf does not activate expression of Sox17, Mixer or Milk. Thus, Xenf is regulated by maternal vegetal positional information in a parallel manner to Sox17 and Mix-related gene pathways. (+info)
Molecular cloning, characterization and evolution of the gene encoding a new group of short-chain alpha-neurotoxins in an Australian elapid, Pseudonaja textilis.
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The structure and organization of five genes responsible for the synthesis of six isoforms of short-chain alpha-neurotoxins in Pseudonaja textilis venom are reported in this paper. This also forms the first report which describes the synthesis of two neurotoxin mRNA variants from one of these genes (Pt-sntx1) as a result of alternative splicing. Each gene consists of three exons which are separated by two introns and each has a functional promoter. The promoter activity was confirmed by both CAT assay and Real-Time PCR. A transcription initiation site, two putative TATA boxes, one CCAAT box and the transcription factor binding consensus sites for AP-1, GATA-2, c/EBPb were identified in the 5' non-coding region of each gene. Phylogenetic analysis showed that these five genes from P. textilis constituted a distinct group which has evolved by gene duplication followed by accelerated evolution from an ancestral gene. (+info)
Interaction of the neurotoxic and nontoxic secretory phospholipases A2 with the crotoxin inhibitor from Crotalus serum.
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Crotalus durissus terrificus snakes possess a protein in their blood, named crotoxin inhibitor from Crotalus serum (CICS), which protects them against crotoxin, the main toxin of their venom. CICS neutralizes the lethal potency of crotoxin and inhibits its phospholipase A2 (PLA2) activity. The aim of the present study is to investigate the specificity of CICS towards snake venom neurotoxic PLA2s (beta-neurotoxins) and nontoxic mammalian PLA2s. This investigation shows that CICS does not affect the enzymatic activity of pancreatic and nonpancreatic PLA2s, bee venom PLA2 and Elapidae beta-neurotoxins but strongly inhibits the PLA2 activity of Viperidae beta-neurotoxins. Surface plasmon resonance and PAGE studies further demonstrated that CICS makes complexes with monomeric and multimeric Viperidae beta-neurotoxins but does not interact with nontoxic PLA2s. In the case of dimeric beta-neurotoxins from Viperidae venoms (crotoxin, Mojave toxin and CbICbII), which are made by the noncovalent association of a PLA2 with a nonenzymatic subunit, CICS does not react with the noncatalytic subunit, instead it binds tightly to the PLA2 subunit and induces the dissociation of the heterocomplex. In vitro assays performed with Torpedo synaptosomes showed a protective action of CICS against Viperidae beta-neurotoxins but not against other PLA2 neurotoxins, on primary and evoked liberation of acetylcholine. In conclusion, CICS is a specific PLA2 inhibitor of the beta-neurotoxins from the Viperidae family. (+info)
Purification and properties of three new phospholipase A2 isoenzymes from Micropechis ikaheka venom.
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Three new phospholipase A2 (PLA2) isoenzymes were purified from the Micropechis ikaheka venom by successive chromatographies. The homogeneity of them was accessed by capillary zone electrophoresis and mass spectrometry. Their N-terminal sequences showed high identity (94, 88 and 90, respectively) with MiPLA-1, a group IB PLA2 also from this venom. In addition, strong immuno-cross-reaction with anti-MiPLA-1 serum was observed. These results suggested that three newly purified PLA2 belonged to group IB. Beside enzymatic activity, they induced various pharmacological effects, including myotoxic, anticoagulant effects and insulin secretion stimulating effects. Our results indicated that enzymatic activity is essential for their myotoxic and anticoagulant effects. On the other hand, no direct correlation between their insulin secretion stimulating effect and enzymatic activity was observed, suggesting that they may stimulate insulin secretion through a non-enzymatic mechanism. (+info)
Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper. A new member of the VEGF family of proteins.
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During the generation of abundant expressed sequence tags from the Viperidae snake Bothrops insularis venom glands, we identified for the first time a cDNA coding for a putative vascular endothelial growth factor-like (VEGF-like) protein. The deduced primary sequence, after complete sequencing of the longest snake venom VEGF (svVEGF) cDNA, displayed similarity with vertebrate VEGFs and with the hypotensive factor from Vipera aspis venom. Its cDNA was subcloned, expressed in Escherichia coli with a His(6) tag as an insoluble monomer, and purified by Ni(2+)-affinity chromatography after 8 m urea extraction. Antiserum against svVEGF was generated and tested in Western blot against proteins from snake venoms and cellular extracts. The mature svVEGF appears to be ubiquitously distributed throughout snake venoms and was also confirmed by Northern blot studies of other related Viperidae species and by cDNA cloning of svVEGF from Bothrops jararaca pit viper. The produced recombinant protein dimerizes after refolding processes and was biologically characterized, showing ability to increase vascular permeability. These results established that svVEGF is a novel and important active toxin during the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins. (+info)