Acute phase proteins: a potential approach for diagnosing chronic infection by Trypanosoma vivax

O objetivo do presente estudo foi verificar possíveis alterações nas proteínas de fase aguda em ovinos infectados experimentalmente com Trypanosoma vivax. Para tanto, foram utilizados oito ovinos machos, sendo quatro usados como controle e quatro infectados com 10(5) tripomastigotas de T. vivax. Colheram-se amostras de sangue em dois tempos antes da infecção e, posteriormente, aos 5, 7, 9, 11, 13, 15, 20, 30, 45, 60, 75, 90, 105 e 120 dias após a infecção (dpi); após centrifugação e aliquotização das amostras. As proteínas de fase aguda foram separadas por eletroforese em gel de acrilamida, contendo dodecil sulfato de sódio, e suas concentrações foram determinadas através de densitometria computadorizada. A dosagem de proteína total foi realizada pelo método colorimétrico do biureto. A contagem dos tripanossomas foi realizada diariamente, utilizando-se uma alíquota de 5 µL de sangue disperso em lâmina de microscopia, sob lamínula de 22 × 22 mm, contando-se os parasitos em 100 campos microscópicos, com objetiva de 40×, multiplicados pelo fator de correção do microscópio, e o resultado expresso em parasitos por mL de sangue. Para a análise estatística, empregou-se o teste de Wilcoxon a 5% de probabilidade. Foi observada a diminuição de diversas proteínas de fase aguda e aumento de antitripsina e transferrina que podem ser utilizadas para auxiliar no diagnóstico da infecção por T. vivax, principalmente na fase crônica da infecção.

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 µM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Telomere biology of trypanosomatids: beginning to answer some questions

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organ ism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

Automated Nuclear Analysis of Leishmania major Telomeric Clusters Reveals Changes in Their Organization during the Parasite's Life Cycle

Parasite virulence genes are usually associated with telomeres. The clustering of the telomeres, together with their particular spatial distribution in the nucleus of human parasites such as Plasmodium falciparum and Trypanosoma brucei, has been suggested to play a role in facilitating ectopic recombination and in the emergence of new antigenic variants. Leishmania parasites, as well as other trypanosomes, have unusual gene expression characteristics, such as polycistronic and constitutive transcription of protein-coding genes. Leishmania subtelomeric regions are even more unique because unlike these regions in other trypanosomes they are devoid of virulence genes. Given these peculiarities of Leishmania, we sought to investigate how telomeres are organized in the nucleus of Leishmania major parasites at both the human and insect stages of their life cycle. We developed a new automated and precise method for identifying telomere position in the three-dimensional space of the nucleus, and we found that the telomeres are organized in clusters present in similar numbers in both the human and insect stages. While the number of clusters remained the same, their distribution differed between the two stages. The telomeric clusters were found more concentrated near the center of the nucleus in the human stage than in the insect stage suggesting reorganization during the parasite's differentiation process between the two hosts. These data provide the first 3D analysis of Leishmania telomere organization. The possible biological implications of these findings are discussed.

Doença de Chagas aguda pós-transfusional sem miocardite

Trata-se de paciente do sexo feminino, com 59 anos de idade, procedente de Itaporanga (SP), diabética e nefropata crônica, internada em virtude de surtos de pielonefnte e insuficiência renal aguda. Dentre outras medidas terapêuticas, recebeu transfusão de sangue. Cerca de dois dias após a última transfusão (sangue oriundo de doador, posteriormente identificado como chagásico) encontraram-se formas tripomastigotas de Trypanosoma cruzi em lâmina preparada para execução de hemograma. Iniciou-se tratamento com Benzonidazol. A paciente cursou para, pleuropneumonia e de secreção purulenta cirúrgica isolou-se Klebsiella spp. A septicemia conduziu a paciente ao êxito letal. Nenhuma lesão tecidual foi observada no miocárdio, no sistema nervoso central, adrenal ou nos demais órgãos examinados.

Spliceosomal Proteomics in Trypanosoma brucei Reveal New RNA Splicing Factors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The taxonomic and phylogenetic relationships of Trypanosoma vivax from South America and Africa

The taxonomic and phylogenetic relationships of Trypanosoma vivax are controversial. It is generally suggested that South American, and East and West African isolates could be classified as subspecies or species allied to T. vivax. This is the first phylogenetic study to compare South American isolates (Brazil and Venezuela) with West/East African T. vivax isolates. Phylogeny using ribosomal sequences positioned all T. vivax isolates tightly together on the periphery of the clade containing all Salivarian trypanosomes. The same branching of isolates within T. vivax clade was observed in all inferred phylogenies using different data sets of sequences (SSU, SSU plus 5.8S or whole ITS rDNA). T. vivax from Brazil, Venezuela and West Africa (Nigeria) were closely related corroborating the West African origin of South American T. vivax, whereas a large genetic distance separated these isolates from the East African isolate (Kenya) analysed. Brazilian isolates from cattle asymptomatic or showing distinct pathology were highly homogeneous. This study did not disclose significant polymorphism to separate West African and South American isolates into different species/subspecies and indicate that the complexity of T. vivax in Africa and of the whole subgenus Trypanosoma (Duttonella) might be higher than previously believed. © 2006 Cambridge University Press.

Reação de trans-splicing in vitro utilizando extrato nuclear livre de células e sequencia parcial de alfa tubulina de Trypanosoma cruzi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação do potencial tripanocida dos óleos essenciais de Melampodium divaricatum e Hedychium coronarium e dos extratos de Polygonum ferrugineum em cepas de Trypanosoma brucei

Pós-graduação em Biotecnologia - IQ

Infecção experimental em caprinos infectados com Trypanosoma evansi Steel, 1885 (Sarcomastigophora: Trypanosomatidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological and molecular characterization and phylogenetic relationships of a new species of trypanosome in Tapirus terrestris (lowland tapir), Trypanosoma terrestris sp nov., from Atlantic Rainforest of southeastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in trypanosoma cruzi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)