fasciculata. In addition, two kinetoplast-associated proteins of T. cruzi, TcKAP4 and TcKAP6, were Epigenetics inhibitor cloned, expressed and antisera were generated against recombinant proteins. Imunolabeling

assays revealed a differential distribution of TcKAPs in the kinetoplast of distinct developmental stages of the parasite. Methods Cell culture Epimastigote forms of T. cruzi (Dm28c clone) [22] PLK inhibitor were grown in liver infusion tryptose (LIT) medium supplemented with 10% fetal calf serum at 28°C. Bloodstream trypomastigote forms derived from the blood of Swiss mice were used to infect the LLC-MK2 cells. Trypomastigotes were released seven days after infection in the supernatant and purified by centrifugation. Amastigotes were obtained by disruption of the LLC-MK2 cells after four days of infection with trypomastigotes. It is worth mentioning that the amastigotes released after disruption of the cells

are mixed with intermediate forms, which BIIB057 purchase represent a transitional stage between amastigotes and trypomastigotes [20]. DNA extraction DNA was extracted as described by Medina-Acosta and Cross [23]. Genome search for T. cruzi orthologs of CfKAPs The CfKAPs1–4 protein sequences were retrieved from GenBank® [24] and a BLASTp search [25] was performed against all protein sequences

from trypanosomatids with a complete sequenced genome, available in GenBank® (release 169). All hits having an e-value lower than 1e10-5 were selected for further analyses. Sequences that were redundant or did not contain a discernible nine amino acids presequence, suggestive of kinetoplast import, were discarded. Evolutionary Anacetrapib analysis of trypanosomatids KAPs Multiple sequence alignments (MSAs) were produced with the ClustalW software [26] and a phylogenetic analysis was performed using the MrBayes software [27, 28], running in parallel [29] in a 28 nodes cluster, by 20,000,000 generations, with gamma correction (estimated α = 6.675), allowing for invariant sites. A mixed amino acid model was used and the Wag fixed rate model [30] prevailed with a posterior probability of 1.0. MSAs and trees were visualized with the Jalview [31] and TreeView software [32], respectively Cloning and expression of the TcKAP4 and TcKAP6 genes Primers were designed to amplify the entire coding region of these genes from the T. cruzi Dm28c genome.