DNA damage induced by metronidazole in Giardia duodenalis triggers a DNA homologous recombination response.

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 12 h of exposure to 3.2 µM 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.

Gene expression profile regulated by the HPV16 E7 oncoprotein and estradiol in cervical tissue.

The HPV16 E7 oncoprotein and 17ß-estradiol are important factors for the induction of premalignant lesions and cervical cancer. The study of these factors is crucial for a better understanding of cervical tumorigenesis. Here, we assessed the global gene expression profiles induced by the HPV16 E7 oncoprotein and/or 17ß-estradiol in cervical tissue of FvB and K14E7 transgenic mice. We found that the most dramatic changes in gene expression occurred in K14E7 and FvB groups treated with 17ß-estradiol. A large number of differentially expressed genes involved in the immune response were observed in 17ß-estradiol treated groups. The E7 oncoprotein mainly affected the expression of genes involved in cellular metabolism. Our microarray data also identified differentially expressed genes that have not previously been reported in cervical cancer. The identification of genes regulated by E7 and 17ß-estradiol, provides the basis for further studies on their role in cervical carcinogenesis.

Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development.

Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development.

Application of 2III7-3 fractional factorial experimental design to enhance enzymatic activities of Pleurotus ostreatus with high concentrations of polychlorinated biphenyls.

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.

Electroeluting DNA fragments.

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.

Nucleic acid and protein factors involved in Escherichia coli polynucleotide phosphorylase function on RNA.

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.