Felipe Olvera

Entamoeba histolytica genomic organization: identification, structure, and phylogenetic relationship of two serine-threonine protein kinases

The protozoan parasite Entamoeba histolytica is the ethiological agent of the human amebiasis, a major cause of death in developing countries (WHO, 1997). Some potential virulence factors such as the galactose-inhibitable lectin, pore-forming peptides and cysteine proteases have been studied (Leippe et al., 1994; McKerrow et al., 1993; Saffer and Petri, 1991). However, it is hard to assess the role of a protein in pathogenesis without a reverse genetic system. Since gene knockout is not yet possible in E. histolytica, some antisense technologies have been used to block specifically gene expression (Bracha et al., 2000; Stock et al., 2001). Therefore, to develop suitable genetic tools, it is important to understand the genomic organization and cell biology of E. histolytica. Because the entamoebal genome is small in size, it has been suggested that its coding sequences are densely packed (Bhattacharya et al., 2000). Analysis of the genomic organization of some linked genes revealed that they are transcribed in the same direction and present short intergenic regions (between 0.4 and 2.3 kb) (Bruchhaus et al., 1993; Petter et al., 1992). Furthermore, it has been observed that the transcripts of two linked genes overlap over a 40 bp stretch, showing no untranscribed intergenic region between them (Gangopadhyay et al., 1997). Cell cycle regulation and signal transduction are poorly understood in E. histolytica. A gene encoding a protein homologous to the yeast cell division regulator p34 has been cloned (Lohia and Samuelson, 1993), while signal transduction pathways activated by components of the extracellular matrix are currently studied (Meza, 2000). In eukaryotic cells, reversible protein phosphorylation is a predominant strategy used to regulate protein activity. This reversible modification is usually accomplished by the addition of a phosphate group to a specific amino acid residue. The phosphate group is transferred from ATP molecules by protein kinases and taken off by protein phosphatases. Proteins regulated by this mechanism play central roles in various cellular activities such as signal transduction and cell cycle regulation. Depending on the residue that phosphorylates, the protein kinases have been divided in two groups: serine–threonine kinases and tyrosine kinases. A 250–300 amino acid catalytic domain, known as the kinase domain, characterizes this large superfamily. Furthermore, the homology of its members is restricted to this domain (Hanks and Hunter, 1994; Schenk and Snaar-Jagalska, 1999). We previously reported the molecular cloning of the E. histolytica gene encoding the SRP54 homolog (Ramos et al., 1997a). Analysis of its upstream sequence led us to the identification of the proteasome asubunit gene (Ramos et al., 1997b). This finding led us to think about the possibility that some other genes were located near those genes. To address the question of whether other genes were present in the 7 kb genomic clone, whence EhSRP54 was isolated, we have performed an ORF FINDER analysis of the full-length clone sequence. We identified the presence of two different polypeptide-encoding sequences. A BLAST search showed that both deduced protein sequences have significant homology to serine–threonine protein kinases from other organisms. One of the genes was named EhYAK1, since it has homology to the YAK1 protein from Saccharomyces cerevisiae (Garrett and Broach, 1989). The second gene, designated EhPK2, was more similar to Dictyostelium discoideum ‘RAC’ kinases (protein kinases that are related to PKA and PKC) (Burki et al., 1991) and a previously reported E. histolytica ‘RAC’ kinase, PK1 (Que et al., 1993). The upper panel of Fig. 1 schematically represents the gene organization of the 7 kb clone. Analysis of the genomic and complementary DNA sequence showed that both entamoebal kinase-coding genes are interrupted by intron sequences (see below). Hence, EhYAK1 and EhPK2 coding sequences are 1827 and 1044 bp long, respectively. As observed in other E. histolytica genes, along with a low G+C content, they show a high A/T codon bias at the third position (more than 67%) (Bhattacharya et al., 2000). To determine the untranslated regions of the EhYAK1 and EhPK2 genes, RACE, and cDNA sequencing experiments were performed; these techniques have been successfully used for other E. histolytica genes (Ramos et al., 1997a). RACE experiments were performed following standard protocols (Bahring et al., 1994; Frohman et al., 1988). Briefly, a modified cDNA was obtained by retrotranscription using an oligo-dT-adapter primer, total entamoebal RNA and a reverse transcriptase enzyme; then, the product was dGtailed with dGTP and the terminal deoxynucleotidyl transferase. Using this modified cDNA as template, the 50 or 30 end of each gene was amplified by PCR. The RACE products were then subcloned into a Experimental Parasitology 100 (2002) 135–139

Use of irradiated elapid and viperid venoms for antivenom production in small and large animals

This work evaluated the feasibility of using toxoids obtained by gamma radiation in the production of antivenoms in small and large animals. Mixtures of African snake venoms from viperids or elapids were used. The viperid mixture contained the crude venom of five species of the genera Echis and Bitis, while the elapid mixture contained the crude venom of six species of the genera Naja and Dendroaspis. The viperid mixture had an LD50 of 1.25 mg/kg in mice, and the elapid mixture had an LD50 of 0.46 mg/kg. Both viper and elapid aqueous mixtures were subjected to Cobalt-60 gamma irradiation in three physical states: lyophilized, frozen and liquid. Radiation doses ranged from 0.5 to 100 kGy. The LD50s of the lyophilized and frozen mixtures of both viperid and elapid mixtures remained unaltered with radiation doses as high as 100 kGy; nevertheless, in the liquid state, doses of 3.5 and 5.5 kGy reduced the venom toxicity of both the viperid and elapid mixtures to 7.25 mg/kg and 1.74 mg/kg; less toxic by factors of 5.8 and 3.8, respectively. Groups of four rabbits and three horses were immunized with either irradiated or non-irradiated mixtures. In vitro and in vivo analysis of the rabbit and horse sera revealed that neutralizing antibodies were produced against both irradiated (toxoids) and native venom mixtures. None of the animals used in this study, either immunized with native venom or toxoids, developed severe local effects due to the application of venoms mixtures. Gamma-irradiated detoxified venoms mixtures, under well-controlled and studied conditions, could be a practical alternative for the production of polyvalent equine serum with high neutralization potency against snake venoms.