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1.
Mem. Inst. Oswaldo Cruz ; 102(1): 97-106, Feb. 2007. tab, ilus
Artigo em Inglês | LILACS | ID: lil-440625

RESUMO

Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.


Assuntos
Animais , Genoma de Protozoário/genética , RNA Nuclear Pequeno/genética , Trypanosoma cruzi/genética , Southern Blotting , Clonagem Molecular , Reação em Cadeia da Polimerase/métodos , Splicing de RNA
2.
Braz. j. med. biol. res ; 40(1): 33-39, Jan. 2007. ilus
Artigo em Inglês | LILACS | ID: lil-439671

RESUMO

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 æM. 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.


Assuntos
Animais , Nitrofurazona/análogos & derivados , Nitrofurazona/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA de Protozoário/efeitos dos fármacos , Tripanossomicidas/farmacologia , Trypanosoma cruzi/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Splicing de RNA/efeitos dos fármacos , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento
3.
Mem. Inst. Oswaldo Cruz ; 99(6): 617-620, Oct. 2004. ilus
Artigo em Inglês | LILACS | ID: lil-387911

RESUMO

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.


Assuntos
Animais , Splicing de RNA , RNA Mensageiro , RNA de Protozoário , Tripanossomicidas , Trypanosoma cruzi , Permeabilidade da Membrana Celular , Éxons , Íntrons , Fatores de Tempo , Transcrição Gênica
4.
Braz. j. med. biol. res ; 37(1): 37-45, Jan. 2004. ilus, tab
Artigo em Inglês | LILACS | ID: lil-352093

RESUMO

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95 percent of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31 percent reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.


Assuntos
Animais , Humanos , Autoanticorpos , Doença de Chagas , Reações Cruzadas , Ribonucleoproteínas Nucleares Pequenas , Trypanosoma cruzi , Autoanticorpos , Western Blotting , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Células HeLa , Testes de Precipitina
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