Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus.

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 µg/20 g body weight, but it is deadly to insects at doses of 10 µg per animal. The venom causes typical alpha and beta-effects on Na+ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K+ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na+ currents verified here, this venom certainly warrant further investigation.

Molecular cloning and nucleotide sequence analysis of genes from a cDNA library of the scorpion Tityus discrepans.

Tityus discrepans is a Venezuelan scorpion known to cause severe human envenomations. It contains toxins that impair proper ion channels function, affect coagulation pathways and interfere with the immunological system, leading to a widespread inflammatory syndrome. This communication reports the results of genes cloned from a cDNA expression library of venomous glands from T. discrepans. A full-length cDNA phagemid library was prepared from which 127 genes were cloned and grouped in 22 clusters showing more than one EST (expressed sequence tag) (74%), and 29 singlets (26%). The identified putative proteins were assorted into two groups. One conformed by precursors similar to gene products implicated in common cellular processes, accounting for 13.4% of transcripts and other comprising putative toxins, representing 50% of total ESTs. A total of 14 sequences are thought to be peptides that recognize or affect Na(+)-channel function and 6 peptides that affect K(+)-channels. Among these two classes of venom components are several for which the peptides were previously isolated and characterized. However, based on sequence similarities, three distinct classes of peptides were also identified and are reported: a bradykinin-potentiating peptide, a defensin-like peptide and an acidic peptide of unknown function. The N-terminal amino acid sequence of several peptides is reported here for the first time. A phylogenetic tree analysis is also reported, as well as three three-dimensional models of representative toxins.

A cDNA for nuclear-encoded chloroplast translational initiation factor 2 from a higher plant is able to complement an infB Escherichia coli null mutant.

Formation of the initiation translation complex containing the three initiation factors, IF1, IF2, and IF3, tRNA(fMet), and GTP constitutes the earliest event in the protein synthesis. IF2, a GTP-binding protein, is the principal factor involved in selecting and binding fMet-tRNA(fMet) to the 30 S ribosomal subunit. Although some chloroplast initiation translational factors have been identified and purified from algae, none of these factors have been characterized from plants. In this work, we report the molecular characterization of a nuclear-encoded chloroplastic IF2 gene from common bean (PvIF2cp). We show that the PvIF2cp gene encodes a protein containing a chloroplast translocation signal peptide, able to target a green fluorescent protein fusion protein to chloroplasts. A high accumulation of PvIF2cp transcript was found in photosynthetic tissues, whereas low mRNA levels were detected in etiolated plants and in nonphotosynthetic organs. Additional data indicate that the PvIF2cp transcript accumulation is modulated by light. The PvIF2cp gene encodes a functional factor, since the PvIF2cp conserved region, containing the G-domain and the C-terminal end, complements an Escherichia coli infB null mutation. Phylogenetic analysis using the PvIF2cp conserved region suggests that the PvIF2cp gene originated via endosymbiotic gene transfer to the nucleus and that it may be a useful marker for phylogeny reconstruction.

Two bean cell wall proteins more abundant during water deficit are high in proline and interact with a plasma membrane protein.

Two antigenically related glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell wall in response to water deficit in Phaseolus vulgaris. In this report, we show that p33 and p36 are able to adhere to leaf protoplasts, and that they bind to plasma membrane (PM) vesicles in a divalent cation-dependent manner. Data from the partial amino acid sequence of the p33 and p36 proteins indicate that they contain repeats of the decapeptide POVYKPOVEK; therefore, they are related to proline-rich proteins. Binding assays demonstrate that both proteins specifically bind to an 80 kDa PM protein. This binding is competed with a peptide that contains the RGD motif, as well as with fibronectin, which also includes this sequence, suggesting that the 80 kDa PM protein has an integrin-like function whose natural ligands are p33 and p36. This is the first case where a PM ligand for a higher plant cell wall protein has been identified.