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Texto completo SciELO Brasil
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Id: biblio-841772
Autor: Villela, Anne Drumond; Rodrigues Junior, Valnês da Silva; Pinto, Antônio Frederico Michel; Wink, Priscila Lamb; Sánchez-Quitian, Zilpa Adriana; Petersen, Guilherme Oliveira; Campos, Maria Martha; Basso, Luiz Augusto; Santos, Diógenes Santiago.
Título: Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis
Fonte: Mem. Inst. Oswaldo Cruz;112(3):203-208, Mar. 2017. graf.
Idioma: en.
Projeto: CNPq; . CNPq; . CNPq; . CNPq; . BNDES; . FAPERGS-CAPES; . CNPq.
Resumo: BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.
Descritores: Macrófagos/microbiologia
Mycobacterium tuberculosis/crescimento & desenvolvimento
Mycobacterium tuberculosis/enzimologia
Mycobacterium tuberculosis/genética
N-Glicosil Hidrolases/genética
-Técnicas de Inativação de Genes
Genes Bacterianos
Limites: Humanos
Responsável: BR1.1 - BIREME

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Id: lil-241598
Autor: Pádula, M.; Boiteux, S..
Título: Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae
Fonte: Braz. j. med. biol. res = Rev. bras. pesqui. méd. biol;32(9):1063-71, Sept. 1999.
Idioma: en.
Resumo: In the present study, we analyzed DNA damage induced by phycocyanin (PHY) in the presence of visible light (VL) using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae
Descritores: Dano ao DNA
Reparo do DNA
DNA/efeitos da radiação
Fármacos Fotossensibilizantes/farmacologia
Saccharomyces cerevisiae/efeitos dos fármacos
-Meios de Cultura
N-Glicosil Hidrolases/fisiologia
Ficocianina/uso terapêutico
Lesões por Radiação
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME

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