In a collaborative work carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG), a polymerase chain reaction multiplex was optimized in order to type ten X-chromosome short tandem repeats (STRs) in a single reaction, including: DXS8378, DXS9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7133, GATA172D05, GATA31E08, and DXS7423. Using this X-decaplex, each 17 of the participating laboratories typed a population sample of approximately 200 unrelated individuals (100 males and 100 females). In this work, we report the allele frequencies for the ten X-STRs in 15 samples from Argentina (Buenos Aires, Córdoba, Río Negro, Entre Ríos, and Misiones), Brazil (São Paulo, Rio de Janeiro, Paraná, and Mato Grosso do Sul), Colombia (Antioquia), Costa Rica, Portugal (Northern and Central regions), and Spain (Galicia and Cantabria). Gene diversities were calculated for the ten markers in each population and all values were above 56%. The average diversity per locus varied between 66%, for DXS7133, and 82%, for DXS6809. For this set of STRs, a high discrimination power was obtained in all populations, both in males (> or =1 in 5 x 10(5)) and females (> or =1 in 3 x 10(9)), as well as high mean exclusion chance in father/daughter duos (> or =99.953%) and in father/mother/daughter trios (> or =99.999%). Genetic distance analysis showed no significant differences between northern and central Portugal or between the two Spanish samples from Galicia and Cantabria. Inside Brazil, significant differences were found between Rio de Janeiro and the other three populations, as well as between São Paulo and Paraná. For the five Argentinean samples, significant distances were only observed when comparing Misiones with Entre Ríos and with Río Negro, the only two samples that do not differ significantly from Costa Rica. Antioquia differed from all other samples, except the one from Río Negro.

In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans-and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.Trypanosomatid parasites utilize RNA splicing for the maturation of nuclear pre-mRNA in two distinct ways: first, as in other eukaryotes, cis splicing is used for intron removal. While this was unambiguously demonstrated for the Trypanosoma brucei poly(A) polymerase (PAP) mRNA (19), intron removal appears to be a rare event in trypanosomatids because a survey of the Tritryp genomes has identified only three additional putative intron-containing genes (10). Second, trypanosomatids process all of their nuclear pre-mRNAs by spliced leader (SL) trans splicing (reviewed in reference 15). In this splicing reaction, the capped, 39 nucleotide (nt)-long 5Ј terminus of the SL RNA, the SL or miniexon, is fused to the 5Ј end of each mRNA. This process is therefore a posttranscriptional mRNA capping mechanism and, since trypanosomatids transcribe their protein coding genes polycistronically, it resolves individual mRNAs from polycistronic precursors in conjunction with polyadenylation. SL trans splicing occurs in several organisms, including tunicate chordates, nematodes, and trematodes, but it is not found in insect and mammalian cells; thus, it is specific to the parasites and not present in the hosts of trypanosomatids. Hence, factors with specific fun...

No abstract

We describe herein an evaluation of the trypanocidal effect of eight piperamides (1-8) isolated from Piper tuberculatum bearing dihydropyridone, piperidine, and isobutyl moieties against epimastigote forms of Trypanosoma cruzi, the causative agent of Chagas' disease. Based on such results, three hydrogenated and two hydrolyzed derivatives (10-14) were prepared and evaluated as well. The dihydropyridone amides (1-3) displayed higher anti-trypanosomal activity. The (Z)-piplartine (1) showed higher activity with a 50% inhibition concentration (IC 50 ) value of 10.5 lM, almost four times more potent than the positive control, benznidazole (IC 50 = 42.7 lM), and should be further evaluated as a suitable hit for the design of new antiprotozoal agents.

Multiple fragments of mitochondrial DNA genes (cytochrome b, cytochrome oxidase I, and 16S rDNA) were used to evaluate the phylogenetic relationships among Triatoma melanocephala, Triatoma tibiamaculata, Triatoma vitticeps, and other members of Triatoma brasiliensis subcomplex under a Bayesian framework and maximum parsimony criterion. With the addition of new sequences of T. tibiamaculata and T. vitticeps, Triatoma juazeirensis, Triatoma melanica and the newly sequenced T. melanocephala, the three first sylvatic species, T. melanocephala, T. tibiamaculata and T. vitticeps, were strongly recovered into a clade separate from the other with the remaining Triatoma species from South America, such as the members of T. brasiliensis subcomplex. Panstrongylus megistus was recovered as a sister to T. tibiamaculata, whereas T. vitticeps was a sister to T. melanocephala. This study revealed the non-monophyly of the T. brasiliensis subcomplex, and the polyphyly of Triatoma was reinforced by the placement of these three sylvatic species with Dipetalogaster, Meccus, Mepraia, and Panstrongylus. The results herein shown highlight the need of generic revision in Triatomini.

The flagellate protozoan Trypanosoma cruzi, the etiological agent of Chagas disease, affects more than 18 million people in Latin America and is responsible for approximately 400000 deaths per year.1) Only two drugs are commercially available for the treatment of this disease, namely, nifurtimox (a 2-nitrofuran derivative) and benznidazole (a 2-nitroimidazole acetamide). These drugs are, however, not consistently effective and, moreover, exhibit serious side effects including cardiac and/or renal toxicity.2) There is thus particular interest in the discovery of natural products that might be developed to generate safer and more efficient chemotherapeutic agents against T. cruzi. 3)A number of biologically-active chromenes have been isolated from species of the genus Piper, including the prenylated chromene 1 from Piper gaudichaudianum 4) and chromenes 2-5 ( Fig. 1) from P. aduncum. 5) Whilst these chromenes have been shown to exhibit anti-fungal 4) and antitumour properties, 5) no evaluation has yet been made with respect to their activity against T. cruzi. The object of the present study was to examine a range of natural chromenes and chromene derivatives in order to determine their potential for further development to treat Chagas disease. METHODS AND RESULTSGeneral All reagents were of analytical grade. For methylation reactions, an ethereal solution of diazomethane (30 ml) was prepared by dissolving 2.14 g of N-methyl-N-nitroso-p-toluenesulphonamide (Diazald; Aldrich, Steinheim, Germany) in 10.0 ml of ethanol containing 4.0% potassium hydroxide.6) Hydrogenation reactions were carried out under an atmosphere of hydrogen in the presence of palladium (3.0 or 10.0%) on activated charcoal (Acros Organics, New Jersey, U.S.A.) as catalyst.7) The acetylated chromene was prepared by treatment with acetic anhydride (20.0 ml) and pyridine (20.0 ml) overnight. 8) Benznidazole, employed as positive control in the assays of trypanocidal activity, was obtained from Roche (Rio de Janeiro, Brazil).Plant Material Specimens of P. gaudichaudianum and P. aduncum were cultivated from seed under greenhouse conditions at the Institute of Chemistry, UNESP, Araraquara-SP, Brazil. Plant material was authenticated by Dr. Guillermo E. D. Paredes (Universidad Pedro Ruiz Gallo, Lambayeque, Peru) and voucher specimens (with codes Kato 0093 and 0057, respectively) were deposited at the Herbarium of the The aim of the study was to investigate the anti-trypanocidal activities of natural chromene and chromene derivatives. Five chromenes were isolated from Piper gaudichaudianum and P. aduncum, and a further seven derivatives were prepared using standard reduction, methylation and acetylation procedures. These compounds were assayed in vitro against epimastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. The results showed that the most of the compounds, especially those possessing electron-donating groups as substituents on the aromatic ring, showed potent trypanocidal activity. The most active compound, [(2S)-methyl-2-m...

RESUMO: "Atividade tripanocida dePiper arboreum e Piper tuberculatum (Piperaceae)". No escopo de nossas pesquisas sobre agentes bioativos da flora brasileira, vinte e quatro extratos e frações de Piper arboreum Aub. e Piper tuberculatum Jacq. (Piperaceae) tiveram sua atividade tripanocida avaliada através do ensaio colorimétrico com MTT. As atividades mais potentes foram manifestadas pelas frações hexânicas das folhas de P. arboreum (CI 50 = 13,3 µg/mL) e P. tuberculatum (CI 50 = 17,2 µg/mL). As frações hexânicas dos frutos de P. tuberculatum e P. arboreum também apresentaram efeito tóxico potente contra as formas epimastigotas de Trypanosoma cruzi, com valores de CI 50 (µg/mL) de 32,2 e 31,3, respectivamente. Adicionalmente, o estudo fitoquímico da fração hexânica das folhas de P. arboreum forneceu duas amidas pirrolidínicas, piperilina (1) e 4,5-diidropiperilina (2), que podem ser responsáveis pela atividade antiprotozoária desta fração.Unitermos: Antiprotozoário, tripanocida, Piper arboreum, Piper tuberculatum, Piperaceae, Trypanosoma cruzi. ABSTRACT:In the scope of our ongoing research on bioactive agents from Brazilian flora, twenty-four extracts and fractions obtained from Piper arboreum Aub. and Piper tuberculatum Jacq. (Piperaceae) were screened for trypanocidal activity by using MTT colorimetric assay. The strongest activity was found in hexane fractions from the leaves of P. arboreum (IC 50 = 13.3 µg/ mL) and P. tuberculatum (IC 50 = 17.2 µg/mL). Hexane fractions of the fruits of P. tuberculatum and P. arboreum showed potent toxic effects on epimastigote forms of Trypanosoma cruzi, with values of IC 50 (µg/mL) of 32.2 and 31.3, respectively. Additionally, the phytochemical study of the hexane fraction of P. arboreum leaves furnished two pyrrolidine amides, piperyline (1) and 4,5-dihydropiperyline (2), which could be responsible, at least in part for the observed antiprotozoal activity.

Abstract. The phylogenetic position of Triatoma sherlocki within triatomines group was inferred by analyzing mtDNA fragments of Cyt B and 16S ribosomal RNA by using maximum parsimony and Bayesian analysis. Despite being differentiated from members of the T. brasiliensis complex on morphologic grounds, molecular phylogenetic analysis suggests T. sherlocki is a member of this complex; moreover, it was placed as a sister species of T. melanica . These suggestions were supported by robust credibility rates. Hence, we show evidence for the paraphyletic group of the " Triatoma brasiliensis complex," which should be composed of T. brasiliensis brasiliensis , T. brasiliensis macromelasoma , T. juazeirensis , T. melanica , and T. sherlocki .