160 resultados para Molecular techniques


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

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

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

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

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation. including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I. a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-1 metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases. (C) 2006 Elsevier B.V. All rights reserved.

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The objective of the present study was to evaluate different techniques for the detection of Paracoccidioides brasiliensis in soil, e.g., culture, animal inoculation and specific DNA amplification by Nested PCR. We designed species-specific inner primers derived from rDNA regions (ITS, 5.8S gene) and found their sensitivity to be higher than culture and animal inoculation. In addition, the sensitivity of these primers was higher than p27-gene primers developed for detection of P brasiliensis in soil in a previous study. DNA from P brasiliensis was detected in soil artificially seeded with the fungus (positive soil control) and from environmental samples collected in an armadillo burrow.

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

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

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While many members of the black yeasts genus Cladophialophora have been reported to cause diseases in humans, understanding of their natural niche is frequently lacking. Some species can be recovered from the natural environment by means of selective isolation techniques. The present study focuses on a Cladophialophora strain that caused an interdigital tinea nigra-like lesion in a HIV-positive Brazilian child. The fungal infection was successfully treated with oxiconazole. Similar strains had been recovered from the environment in Brazil, Uruguay and the Netherlands. The strains were characterized by sequencing the Internal Transcribed Spacer (ITS) regions and the small subunit (SSU) of the nuclear ribosomal RNA gene, as well as the elongation factor 1-alpha (EF1) gene. Since no match with any known species was found, it is described as the new species, Cladophialophora saturnica.

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A variante de hemoglobina (Hb) D mais comum, Hb D Los Angeles ou D Punjab, é originada de uma transversão GAA->CAA no códon 121 da globina beta; essa mutação resulta na substituição do ácido glutâmico por glutamina na proteína. É a terceira variante de hemoglobina mais freqüente da população brasileira. Como as hemoglobinas D apresentam migração similar à hemoglobina S em pH alcalino, e com a hemoglobina A em pH ácido, são necessários vários testes para o correto diagnóstico. No presente estudo objetivou-se relacionar os diferentes procedimentos laboratoriais de rotina diagnóstica, além da análise molecular, para estabelecer o perfil de Hb D Los Angeles no Brasil. Foram analisados 47 indivíduos da população brasileira com provável Hb D Los Angeles, por vários procedimentos eletroforéticos em diferentes condições de pH, além da cromatografia líquida de alta pressão, e testes moleculares para confirmação da mutação. Foram encontrados quatro tipos de combinações de hemoglobinas: 42 indivíduos portadores de hemoglobina AD Los Angeles, dois indivíduos com doença de Hb S/D Los Angeles, dois indivíduos com Hb D Los Angeles e talassemia beta e um indivíduo com Hb D Los Angeles e Hb Lepore. Os indivíduos heterozigotos para D Los Angeles são assintomáticos, entretanto, em associação com outras variantes e talassemias podem apresentar graus variáveis de manifestações clínicas. Os resultados apresentados enfatizaram a necessidade da associação de várias metodologias para a identificação da Hb D Los Angeles, além de auxiliar na elucidação de combinações raras.

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Opportunistic fungal pathogens are becoming increasingly important causes of both community-acquired and nosocomial infections. The most important fungal pathogens are yeast species belonging to the genus Candida. These species show differences in levels of resistance to antifungal agents and mortality. Consequently, it is important to correctly identify the causative organism to the species level. Identification of Candida dubliniensis in particular remains problematic because of the high degree of phenotypic similarity between this species and Candida albicans. However, as the differences between both are most pronounced at the genetic level, several studies have been conducted in order to provide a specific and rapid identification fingerprinting molecular test. In most candidal infectious, no single DNA fingerprinting technique has evolved as a dominant method, and each method has its advantages, disadvantages and limitations. Moreover, the current challenge of these techniques is to compile standardized patterns in a database for interlaboratory use and future reference. This review provides an overview of most common molecular fingerprinting techniques currently available for discrimination of C. albicans and C. dubliniensis.

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Haemoglobin, the 'honorary enzyme' [Brunori (1999), Trends Biochem. Sci. 24, 158-161], constitutes a prime prototype for allosteric models. Here, the crystallization and preliminary X-ray analysis of haemoglobin I from the South American fish Brycon cephalus are reported. X-ray diffraction data have been collected to 2.5 Angstrom resolution using synchrotron radiation (LNLS). Crystals were determined to belong to the space group P6(1)22 and preliminary structural analysis revealed the presence of one dimer (alpha beta) in the asymmetric unit. The structure was determined using standard molecular-replacement techniques.

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Anaplasmataceae organisms comprise a group of obligate intracellular gram-negative, tick-borne bacteria that can infect both animals and humans. In the present work we investigate the presence of Ehrlichia, Anaplasrna, and Neorickettsia species in blood samples from Brazilian marsh deer (Blastocerus dichotomus), using both molecular and serologic techniques. Blood was collected from 143 deer captured along floodplains of the Parana River, near the Porto Primavera hydroelectric power plant. Before and after flooding, marsh deer were captured for a wide range research program under the financial support of São Paulo State Energy Company (CESP), between 1998 and 2001. Samples were divided into four groups according to time and location of capture and named MS01 (n = 99), MS02 (n = 18) (Mato Grosso do Sul, before and after flooding, respectively), PX (n = 9; Peixe River, after flooding), and AGUA (n = 17; Aguapei River, after flooding). The seroprevalences for Ehrlichia chaffeensis and Anaplasma phagocytophilum were 76.76% and 20.2% in MS01, 88.88% and 5.55% in MS02, 88.88% and 22.22% in PX, and 94.12% and 5.88% in AGUA, respectively. Sixty-one animals (42.65% of the total population) were PCR-positive for E. chaffeensis PCR (100.0% identity based on 16S rRNA, dsb, and groESL genes). Seventy deer (48.95% of the total population) were PCR-positive for Anaplasma spp. (99.0% of identity with A. platys, and in the same clade as A. phagocytophilum, A. bovis, and A. platys based on 16S rRNA phylogenetic analysis). Our results demonstrate that Brazilian marsh deer are exposed to E. chaffeensis and Anaplasma spp. and may act as reservoirs for these rickettsial agents, playing a role in disease transmission to humans and other animals. (c) 2012 Elsevier Ltd. All rights reserved.