RESUMO
Standard diagnostic methods currently in use for the identification of helminth infections in ruminants are based on the morphological analysis of immature and adult stages of parasites. This paper describes a method for the semiquantitative identification of nematodes, mainly Trichostrongyloidea, at species-level resolution. The method is based on amplification and fragment analysis followed by minisequencing of the ITS-2 region (internal transcribed spacer 2) of the ribosomal DNA of parasite eggs or larvae. This method allows for the identification of seven genera (Chabertia, Cooperia, Haemonchus, Oesophagostomum, Ostertagia, Teladorsagia, and Trichostrongylus) and 12 species (Chabertia ovina, Cooperia curticei, Cooperia punctata, Cooperia oncophora/Cooperia surnabada, Haemonchus contortus, Haemonchus placei, Haemonchus longistipes, Oesophagostomum asperum, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei, and Trichostrongylus colubriformis) of infectious nematodes of domestic ruminants. The concordance between the morphological and molecular analyses in the detection of genera ranged from 0.84 to 0.99, suggesting the proposed detection method is specific, semiquantitative, less laborious, and highly cost-efficient.
Assuntos
Infecções por Nematoides/veterinária , Ruminantes/parasitologia , Trichostrongyloidea/isolamento & purificação , Animais , Bovinos , Doenças dos Bovinos/parasitologia , DNA de Helmintos , DNA Ribossômico , Cabras , Haemonchus/genética , Haemonchus/isolamento & purificação , Infecções por Nematoides/parasitologia , Técnicas de Amplificação de Ácido Nucleico/veterinária , Oesophagostomum/genética , Oesophagostomum/isolamento & purificação , Ostertagia/genética , Ostertagia/isolamento & purificação , Ovinos , Strongyloidea/genética , Trichostrongyloidea/genética , Trichostrongylus/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Quina is a popular name originally attributed to Cinchona pubescens Vahl (=Cinchona succirubra) and Cinchona. calisaya Wedd., species native from Peru that have the antimalarial alkaloid quinine. In Brazil, bitter barks substitutes for the Peruvian species began to be used centuries ago, and they still are sold in popular markets. To assess the authenticity and the conditions on which samples of quinas have been commercialized, using the DNA barcode, chemical and biological assays. MATERIALS AND METHODS: Starting with 28 samples of barks acquired on a popular market, 23 had their DNA extracted successfully. The regions matK and rbcL were amplified and sequenced for 15 and 23 samples, respectively. Phytochemical analyses were performed by chromatographic methods, and biological essays were done by antimalarial tests in vitro. RESULTS: The identified species belonged to six different families, many of them endangered or with no correlation with use in traditional medicine as a Brazilian quina. The absence of typical bitter chemical substances indicated that barks have been collected from other species or from very young trees. The results of biological essays confirm the lack of standardization of the sold materials. CONCLUSION: The integrated approaches proved to be efficient to evaluate medicinal plants sold in popular markets and can be useful for promoting their better use and conservation.
Assuntos
Cinchona/química , Conservação dos Recursos Naturais , Medicina Tradicional/métodos , Plantas Medicinais/química , Antimaláricos/química , Antimaláricos/economia , Antimaláricos/isolamento & purificação , Sequência de Bases , Brasil , Cinchona/genética , Comércio , Código de Barras de DNA Taxonômico , Etnofarmacologia , Humanos , Medicina Tradicional/economia , Casca de Planta , Extratos Vegetais/química , Extratos Vegetais/economia , Extratos Vegetais/farmacologia , Plantas Medicinais/genéticaRESUMO
Nucleotide excision repair (NER) is a highly conserved genome repair pathway acting on helix distorting DNA lesions. NER is divided into two subpathways: global genome NER (GG-NER), which is responsible for repair throughout genomes, and transcription-coupled NER (TC-NER), which acts on lesions that impede transcription. The extent of the Trypanosoma brucei genome that is transcribed is highly unusual, since most genes are organized in multigene transcription units, each transcribed from a single promoter. Given this transcription organization, we have addressed the importance of NER to T. brucei genome maintenance by performing RNAi against all predicted contributing factors. Our results indicate that TC-NER is the main pathway of NER repair, but only CSB, XPBz and XPG contribute. Moreover, we show that UV lesions are inefficiently repaired in T. brucei, perhaps due to preferential use of RNA polymerase translesion synthesis. RNAi of XPC and DDB was found to be lethal, and we show that these factors act in inter-strand cross-link repair. XPD and XPB appear only to act in transcription, not repair. This work indicates that the predominance of multigenic transcription in T. brucei has resulted in pronounced adaptation of NER relative to the host and may be an attractive drug target.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Transcrição Gênica , Trypanosoma brucei brucei/fisiologia , Enzimas Reparadoras do DNA/genética , Genes Essenciais , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
SMYB1 is a Schistosoma mansoni protein highly similar to members of the Y-box binding protein family. Similar to other homologues, SMYB1 is able to bind double- and single-stranded DNA, as well as RNA molecules. The characterization of proteins involved in the regulation of gene expression in S. mansoni is of great importance for the understanding of molecular events that control morphological and physiological changes in this parasite. Here we demonstrate that SMYB1 is located in the cytoplasm of cells from different life-cycle stages of S. mansoni, suggesting that this protein is probably acting in mRNA metabolism in the cytoplasm and corroborating previous findings from our group that showed its ability to bind RNA. Protein-protein interactions are important events in all biological processes, since most proteins execute their functions through large supramolecular structures. Yeast two-hybrid screenings using SMYB1 as bait identified a partner in S. mansoni similar to the SmD3 protein of Drosophila melanogaster (SmRNP), which is important in the assembly of small nuclear ribonucleoprotein complexes. Also, pull-down assays were conducted using immobilized GST-SMYB1 proteins and confirmed the SMYB1-SmRNP interaction. The interaction of SMYB1 with a protein involved in mRNA processing suggests that it may act in processes such as turnover, transport and stabilization of RNA molecules.
Assuntos
Proteínas de Helminto/metabolismo , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismo , Schistosoma mansoni/metabolismo , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Transporte Biológico , Citoplasma/metabolismo , Feminino , Biblioteca Gênica , Proteínas de Helminto/genética , Imuno-Histoquímica , Masculino , RNA de Helmintos/genética , RNA Mensageiro/genética , Coelhos , Schistosoma mansoni/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Parasitic nematodes of the genus Haemonchus infect a range of ruminant hosts and are of major veterinary and economic importance. In this study, the genetic variability of seven isolates of Haemonchus placei and Haemonchus contortus was evaluated using the mitochondrial gene cytochrome oxidase subunit I and the nuclear gene b-tubulin isotype 1. A total of 156 specimens were obtained from cattle, sheep, goat and buffalo herds raised on commercial properties from the southern and southeastern regions of Brazil and identified to the species level by sequencing of the nuclear internal transcribed spacer 2. Thirty-four percent of the specimens were identified as H. placei and 66% as H. contortus. Cattle were the preferred hosts for H. placei, whereas H. contortus was most frequent in the other three ruminant species. Analysis of genetic differentiation between isolates revealed that high rates of gene flow are operating among populations of both nematode species, including among those from different ruminant host species. Populations of H. placei were less polymorphic and presented a lower frequency of single nucleotide polymorphisms associated with benzimidazole resistance compared with H. contortus. In line with the low amount of genetic structure observed among isolates, alleles of b-tubulin 1 associated with benzimidazole resistance were present at relatively high frequencies of 520% in isolates of H. contortus from farms that never used this class of anthelmintic. The results presented here are consistent with the hypothesis of multiple origins of alleles associated with benzimidazole resistance, with the trade of animals among properties acting as the main factor promoting the spread of anthelmintic resistance.
Assuntos
Animais Domésticos/parasitologia , Variação Genética , Haemonchus/classificação , Haemonchus/genética , Ruminantes/parasitologia , Animais , Brasil , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Fluxo Gênico , Haemonchus/isolamento & purificação , Masculino , Dados de Sequência Molecular , Recombinação Genética , Análise de Sequência de DNARESUMO
BACKGROUND: During its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite. METHODOLOGY/PRINCIPAL FINDINGS: In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p