RESUMO
PA28γ is a proteasome activator involved in the regulation of the cellular proliferation, differentiation and growth. In the present study, we identified and characterized a cDNA from Schistosoma mansoni exhibiting significant homology to PA28γ of diverse taxa ranging from mammals (including humans) to simple invertebrates. Designated SmPA28γ, this transcript has a 753bp predicted ORF encoding a protein of 250 amino acid residues. Alignment of SmPA28γ with multiple PA28γ orthologues revealed an average similarity of ~40% among the investigated organisms, and 90% similarity with PA28γ from Schistosoma japonicum. In addition, phylogenetic analysis demonstrated a close linkage between SmPA28γ to its sister group that contains well-characterized PA28γ sequences from Drosophila spp., as well as sharing the same branch with PA28γ from S. japonicum. Gene expression profiling of SmPA28γ using real-time quantitative PCR revealed elevated steady-state transcript levels in the eggs, miracidia and paired adult worms compared to other stages. In parallel with gene expression profiles, an affinity-purified anti-SmPA28γ antibody produced against recombinant protein exhibited strongest reactivity in Western blot analyses to endogenous SmPA28γ from miracidia, sporocysts and paired adult worms. Given its known regulatory function in other organisms, we hypothesized that the high level of SmPA28γ transcript and protein in these stages may be correlated with an important role of the PA28γ in the cellular growth and/or development of this parasite. To address this hypothesis, miracidia were transformed in vitro to sporocysts in the presence of SmPA28γ double-stranded RNAs (dsRNAs) and cultivated for 4 days, after which time steady-state transcript and protein levels, and phenotypic changes were evaluated. SmPA28γ dsRNA treatment resulted in gene and protein knockdown of ~60% and ~80%, respectively, which were correlated with a significant decrease in larval length compared to its controls. These findings are consistent with a putative role of SmPA28γ in larval growth/development of the S. mansoni.
Assuntos
Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Proteínas Musculares/genética , Complexo de Endopeptidases do Proteassoma/genética , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/genética , Sequência de Aminoácidos , Animais , Análise por Conglomerados , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The in vitro schistosomicidal activity of curcumin (doses ranging from 5 to 100 microM) was carried out against Schistosoma mansoni adult worms. Curcumin (at 50 and 100 microM) caused death of all worms. When tested at the doses of 5 and 20 microM, it decreased the worm viability in comparison with negative (Roswell Memorial Park Institute (RPMI) 1640 medium alone or RPMI 1640 medium with 10% dimethyl sulfoxide) and positive (heat-killed worms at 56 degrees C or praziquantel 10 microM) control groups. All pairs of coupled adult worms were separated into individual male and female by the action of curcumin at the doses of 20 to 100 microM. When tested at 5 and 10 microM, curcumin reduced egg production by 50% in comparison with the positive control group. It is the first time that the schistosomicidal activity has been reported for curcumin.
