, 1996 and Turk et al., 2000). The activation peptide length varied from 94 to 110 amino acids in insect cathepsin L sequences analyzed in the present study. After cleavage, these peptides act as cathepsin L inhibitors, playing an important role in the activity regulation Stem Cell Compound Library clinical trial of these digestive enzymes ( Coulombe et al., 1996 and Cygler and Mort, 1997). Considering the ERFNIN and GCNGG motifs, important for the globular folding of the N-terminus of the activation peptide ( Coulombe et al., 1996), the T. infestans cathepsin L sequence (ERYNIN, GCDGG) differed from that of T. brasiliensis

and R. prolixus (ERFNIN, GCEGG). The GNFD motif was more variable, modified to KNFD in TBCATL-2 and T. infestans cathepsin L, MNFD in TBCATL-1 and KNLF in the R. prolixus cathepsin L amino acid sequence ( Lopez-Ordoñez et al., 2001 and Kollien et al., 2004). The initial amino acids of the mature enzyme (Leu-Pro), the

number of disulfide bridge forming cysteine residues, the active site and S2 residues were identical in all four triatomine cathepsin L sequences. Both mature T. brasiliensis cathepsin L amino acid sequences had a closer identity with cathepsin L of R. prolixus than that of T. infestans. Therefore the sequence of T. infestans was separated from the other three triatomine cathepsins in the dendrogram. This result indicates the occurrence of, at least, two cathepsin L subgroups in triatomines. T. brasiliensis and T. infestans are phylogenetically closer than T. brasiliensis and R. prolixus, therefore TBCATL-1 and TBCATL-2 should cluster together with the amino acid sequence Alectinib in vivo of T. infestans. Since this is not the case, we can conclude that TBCATL-1/-2 and R. prolixus cathepsin L encoding genes might be orthologous counterparts, whereas the more distant T. infestans cathepsin L belongs to a second triatomine cathepsin L the group. If we include different cathepsin B, cathepsin D, carboxy- and amino-peptidase isoforms, so far identified at DNA and protein level, the complexity of the triatomine digestive system

becomes clearer. Expression analyses by RT-PCR and northern blotting have shown high cathepsin L transcript abundance in the posterior intestine (small intestine) of R. prolixus whereas in the crop (stomach) cathepsin L mRNA was absent ( Lopez-Ordoñez et al., 2001). These authors also have shown high cathepsin L transcript abundance in second instar nymphs, lower in unfed first instar nymphs and in fed first, third and fourth instars nymphs but absent in fifth instars. These findings are surprising as the last nymphal stage is also strongly dependent on blood digestion in view of nutrient demand for the metamorphosis to adults and because in adult R. prolixus, cathepsin L mRNA has been detected by northern blotting. By contrast, in the present study both cathepsin L transcripts were highly abundant in the small intestine of fifth instar nymphs.