RESUMO
The mechanisms underlying metronidazole (MTZ) resistance in Giardia duodenalis have been associated with decreased activity of the enzymes implicated in its activation including nitroductase-1, thioredoxin reductase and pyruvate-ferredoxin oxidoreductase (PFOR). MTZ activation generates radicals that can form adducts with proteins such as thioredoxin reductase and α- and -ß giardins as well as DNA damage resulting in trophozoite's death. The damage induced in DNA requires a straight forward response that may allow parasite survival. Here, we studied changes in histone H2A phosphorylation to evaluate the DNA repair response pathway after induction of double strand break (DSB) by MTZ in Giardia DNA. Our results showed that the DNA repair mechanisms after exposure of Giardia trophozoites to MTZ, involved a homologous recombination pathway. We observed a significant increase in the expression level of proteins GdDMC1B, which carries out Rad51 role in G. duodenalis, and GdMre11, after 12â¯h of exposure to 3.2⯵M MTZ. This increase was concomitant with the generation of DSB in the DNA of trophozoites treated MTZ. Altogether, these results suggest that MTZ-induced DNA damage in Giardia triggers the DNA homologous recombination repair (DHRR) pathway, which may contribute to the parasite survival in the presence of MTZ.
Assuntos
Antiprotozoários/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Giardia lamblia/efeitos dos fármacos , Metronidazol/farmacologia , DNA de Protozoário/efeitos dos fármacos , DNA de Protozoário/genética , Resistência a Medicamentos , Giardia lamblia/genética , Marcação In Situ das Extremidades Cortadas , Concentração Inibidora 50 , Fosforilação/efeitos dos fármacos , Recombinação Genética/genéticaRESUMO
A 2(III)(7-3) fractional factorial experimental design was used to establish 16 culture media, with and without PCBs to enhance the activities of laccase (Lac), manganese peroxidase (MnP), and versatile peroxidase (VP) produced by the white rot fungus Pleurotus ostreatus. The culture was added to 10,000 mg L(-1) of transformer oil, containing 71% of the identified Arochlor 1242. The culture conditions were established with eight variables at two values (levels); pH (4 and 6), agitation (100 and 200 rpm), CuSO(4) (150 and 250 mg L(-1)), MnSO(4) (50 and 200 mg L(-1)), Tween 80 (13 and 3500 mg L(-1)), wheat straw (0 and 2.5 g L(-1)), sugarcane bagasse (0 and 2.5 g L(-1)),and Arochlor 1242 (0 and 7100 mg L(-1)) at 4, 8, 12, 16 and 20 days old culture. Laccase activity was enhanced at a high value of pH and low value of agitation (P<0.001) and correlated positively (R(2)= 0.9; α=0.05) with the removal of polychlorinated biphenyls (PCBs). VP activity was enhanced 27-fold with PCBs, Tween 80 and pH. The MnP activity was increased 1.2-fold with PCBs. The fractional factorial experimental design methodology allowed us to determine the P. ostreatus culture media conditions to enhance Lac and VP activities for efficient removal of Arochlor 1242 (one of the most recalcitrant organochloride pollutants). The factors that shown the greatest effect on Lac activity were: pH, agitation and high concentrations of Arochlor 1242.
Assuntos
Meios de Cultura/química , Poluentes Ambientais/metabolismo , Lacase/metabolismo , Peroxidase/metabolismo , Peroxidases/metabolismo , Pleurotus/enzimologia , Bifenilos Policlorados/metabolismo , Arocloros/metabolismo , Biodegradação Ambiental , Concentração de Íons de Hidrogênio , Modelos Lineares , Modelos Químicos , Fatores de TempoRESUMO
It has been reported that polynucleotide phosphorylase (PNPase) binds to RNA via KH and S1 domains, and at least two main complexes (I and II) have been observed in RNA-binding assays. Here we describe PNPase binding to RNA, the factors involved in this activity and the nature of the interactions observed in vitro. Our results show that RNA length and composition affect PNPase binding, and that PNPase interacts primarily with the 3' end of RNA, forming the complex I-RNA, which contains trimeric units of PNPase. When the 5' end of RNA is blocked by a hybridizing oligonucleotide, the formation of complex II-RNA is inhibited. In addition, PNPase was found to form high molecular weight (>440 kDa) aggregates in vitro in the absence of RNA, which may correspond to the hexameric form of the enzyme. We confirmed that PNPase in vitro RNA binding, degradation and polyadenylation activities depend on the integrity of KH and S1 domains. These results can explain the defective in vivo autoregulation of PNPase71, a KH point substitution mutant. As previously reported, optimal growth of a cold-sensitive strain at 18 degrees C requires a fully active PNPase, however, we show that overexpression of a novel PNPaseDeltaS1 partially compensated the growth impairment of this strain, while PNPase71 showed a minor compensation effect. Finally, we propose a mechanism of PNPase interactions and discuss their implications in PNPase function.