Venom gland transcriptomes of two elapid snakes (Bungarus multicinctus and Naja atra) and evolution of toxin genes

<p>Abstract</p> <p>Background</p> <p>Kraits (genus <it>Bungarus</it>) and cobras (genus <it>Naja</it>) are two representative toxic genera of elapids in the old world. Although they are closely related genera and both of their venoms are very toxic, the compositions of their venoms are very different. To unveil their detailed venoms and their evolutionary patterns, we constructed venom gland cDNA libraries and genomic bacterial artificial chromosome (BAC) libraries for <it>Bungarus multicinctus </it>and <it>Naja atra</it>, respectively. We sequenced about 1500 cDNA clones for each of the venom cDNA libraries and screened BAC libraries of the two snakes by blot analysis using four kinds of toxin probes; <it>i.e</it>., three-finger toxin (3FTx), phospholipase A2 (PLA2), kunitz-type protease inhibitor (Kunitz), and natriuretic peptide (NP).</p> <p>Results</p> <p>In total, 1092 valid expressed sequences tags (ESTs) for <it>B. multicinctus </it>and 1166 ESTs for <it>N. atra </it>were generated. About 70% of these ESTs can be annotated as snake toxin transcripts. 3FTx (64.5%) and <it>β </it>bungarotoxin (25.1%) comprise the main toxin classes in <it>B. multicinctus</it>, while 3FTx (95.8%) is the dominant toxin in <it>N. atra</it>. We also observed several less abundant venom families in <it>B. multicinctus </it>and <it>N. atra</it>, such as PLA2, C-type lectins, and Kunitz. Peculiarly a cluster of NP precursors with tandem NPs was detected in <it>B. multicinctus</it>. A total of 71 positive toxin BAC clones in <it>B. multicinctus </it>and <it>N. atra </it>were identified using four kinds of toxin probes (3FTx, PLA2, Kunitz, and NP), among which 39 3FTx-postive BACs were sequenced to reveal gene structures of 3FTx toxin genes.</p> <p>Conclusions</p> <p>Based on the toxin ESTs and 3FTx gene sequences, the major components of <it>B. multicinctus </it>venom transcriptome are neurotoxins, including long chain alpha neurotoxins (<it>α</it>-ntx) and the recently originated <it>β </it>bungarotoxin, whereas the <it>N. atra </it>venom transcriptome mainly contains 3FTxs with cytotoxicity and neurotoxicity (short chain <it>α</it>-ntx). The data also revealed that tandem duplications contributed the most to the expansion of toxin multigene families. Analysis of nonsynonymous to synonymous nucleotide substitution rate ratios (<it>dN</it>/<it>dS</it>) indicates that not only multigene toxin families but also other less abundant toxins might have been under rapid diversifying evolution.</p

A Family of Diverse Kunitz Inhibitors from Echinococcus granulosus Potentially Involved in Host-Parasite Cross-Talk

The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H+-treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 – EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (KI*) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection

Alternative methods providing enhanced sensitivity and selectivity in capillary electroseparation experiments

The study of alternative and novel techniques for altering selectivity and enhancing sensitivity as well as injection and detection protocols are important in the ongoing development of capillary electroseparation protocols. Some recent research from our laboratory in these fields is presented and discussed in this review. To improve sensitivity an off-line sample enrichment technique utilising solvent evaporation in a levitated drop or an on-line solid-phase extraction protocol was used. The selectivity was tuned by the use of protein gels or molecularly imprinted polymer mediated capillary electrochromatography. Furthermore, a picolitre droplet injection method is described as well as a detection protocol based on laser-induced fluorescence imaging. (C) 2000 Elsevier Science B.V, All rights reserved

Molecularly imprinted capillary electrochromatography for selective determination of thiabendazole in citrus samples

In this work, the suitability of the combination of molecular imprinting and capillary electrochromatography (MIP-CEC) to be used as powerful tool in environmental or food analysis has been for the first time studied and successfully demonstrated. A molecularly imprinted monolith (MIM) has been synthesised and evaluated as stationary phase for the selective determination of the fungicide thiabendazole (TBZ) in citrus samples by non-aqueous capillary electrochromatography. The influence of the mobile phase composition, the voltage of the power supply and the separation temperature on the recognition of TBZ by the imprinted polymer has been evaluated, and the imprint effect in the MIM was clearly demonstrated. Once optimum recognition conditions were established, other variables affecting mechanical properties and chromatographic performance of MIM were adjusted using computational approach. The high selectivity achieved by the MIP-CEC developed procedure allowed unambiguous detection and quantification of TBZ in citrus samples by direct injection of the crude sample extracts, without any previous clean-up, in less than 6 min. The developed method was properly validated and the calculated detection limits were bellow the established maximum residue limits (MRLs), clearly demonstrating the suitability of the method to be used for the control of the selected fungicide