Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3 0 single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR. IntroductionDNA double-strand breaks (DSBs) are the most dangerous DNA damage, as a single unrepaired DSB can trigger apoptosis. DSBs are generated during physiological replication and induced by ionizingradiation. DSBs are repaired by two major DSB-repair pathways, homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice of DSB-repair pathway depends on the cell-cycle phase and the DNA-damaging agent (Symington & Gautier 2011). HR repairs DSBs in the S to G 2 phases, whereas NHEJ operates in all the cell phases. HR is more prominent than NHEJ in the repair of DSBs occurring during DNA replication (Hochegger et al. 2006;Qing et al. 2011) and is essential for cellular proliferation. Indeed, loss of critical HR factors, including CtIP, Mre11 and Rad51, causes mortality due to severe genome instability (Yamazoe et al. 2004;Nakamura et al. 2010;Hoa et al. 2015).HR is carried out in a series of steps, beginning with the 5 0 -to-3 0 strand resection of DSBs, which is called DSB resection (reviewed in Stracker & Petrini 2011;Symington & Gautier 2011). The resulting 3 0 -overhang is coated with a single-strand DNA binding protein, replication protein A (RPA). RPA is subsequently replaced with polymerized Rad51 recombinase, which polymerization results in the formation of subnuclear Rad51 foci. Polymerized Rad51 performs homology search and strand invasion into intact homologous sequences leading to formation of D-loop and Holliday junction structures. Biochemical and genetic studies have shown that in Saccharomyces cerevisiae (S. cerevisiae), DSB resection is initiated by Mre11 nuclease, which physically associates with Rad50 and Xrs2 (the MRX complex). The MRX complex and Sae2 are the orthologs of human Mre11/ Rad50/Nbs1 (the MRN complex) and CtIP, respectively. Yeast MRX ...
Our transgenic (Tg) strain carrying copies of the human c-Ha-ras proto-oncogene is highly susceptible to 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis, possibly due to activation of the transgene, and can be used in medium-term bioassay models to test for modifying effects of estrogenic environmental compounds on tumor development. The present study was conducted to assess the influence of dietary feeding of the endocrine disruptors atrazine and nonylphenol on DMBA-induced carcinogenesis in c-Ha-ras Tg rats. Animals of both sexes were given a single oral dose of DMBA (25 mg/kg body weight) at 50 days of age and thereafter received soybean-free diet containing 5, 50 or 500 ppm atrazine, or 10, 25, 100 or 250 ppm nonylphenol. In female Tg rats, atrazine at a dose of 5 ppm increased the incidences of mammary adenomas and adenocarcinomas (P < < < <0.01 and P < < < <0.05), while 50 ppm increased the adenocarcinoma incidence (P < < < <0.05). In males, skin tumor development, in contrast, was significantly decreased at the highest dose. Nonylphenol at 10 ppm increased adenocarcinoma and total mammary tumor multiplicity in female Tg rats (P < < < <0.05), but there was no dose dependence, a significant quadratic dose-response trend rather being observed (P < < < <0.05). In vitro, atrazine did not cause proliferation of MCF-7 cells at any of a range of doses tested. These results suggest that endocrine disruptors may enhance mammary carcinogenesis, but only in a certain limited dose range under the present experimental conditions. The doses applied, moreover, were all extremely high compared to the possible environmental human exposure levels. (Cancer Sci 2004; 95: 404-410) t is well documented that estrogen is an obligatory factor for development of the mammary gland.1, 2) The hormone also plays an important role in both the etiology and treatment of mammary cancer and accumulating evidence indicates that slightly elevated levels of circulating estrogens may predispose to tumorigenesis.3, 4) Exogenous estrogens may similarly increase mammary cancer risk.5-7) This may be of considerable importance, since recently, a number of environmental chemicals impacting on the endocrine system (endocrine disruptors) have been shown to exhibit estrogenic activity, these including triazine derivatives and alkylphenolic compounds.Among the symmetrical triazine-type herbicides, atrazine (6-chloro-N 2 -ethyl-N 4 -isopropyl-1,3,5-triazine-2,4-diamine) is one of the most widely and heavily used herbicides all over the world. It is employed in agriculture as a selective pre-and postemergence agent for annual control of grass and broad-leaved weeds. A number of studies have suggested that atrazine is an endocrine disruptor [8][9][10][11][12][13][14][15] and in female Sprague-Dawley, but not other strains of rats, high doses (50-1000 ppm) in the diet have been found to be associated with an increased incidence and/or an earlier onset of mammary gland tumors. [16][17][18][19][20] Alkylphenol polyethoxylates are the se...
A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an immunosensor based on surface plasmon resonance (SPR-sensor) were developed for fungicide boscalid determination in horticultural crops. To produce antiboscalid monoclonal antibodies (MoAb BSC7 and MoAb BSC72) for these assays, a hapten of boscalid was synthesized and conjugated to keyhole limpet hemocyanin for Balb/c mouse immunization. The working range of the dc-ELISA was 0.8-16 ng/mL with MoAb BSC7 and 2.5-120 ng/mL with MoAb BSC72, and that of the SPR-sensor was 17-80 ng/mL with MoAb BSC7. The dc-ELISA and SPR-sensor were compared for their sensitivity in determining boscalid residues at the maximum residue limit of 1-40 mg/kg for horticultural crops in Japan. Recovery of the spiked boscalid was 85-109% by the SPR-sensor and 100-124% by the dc-ELISA. On real tomato samples, the results obtained by both of these immunoassays correlated well with the results obtained by high-performance liquid chromatography.
An immunosensor based on surface plasmon resonance (SPR-sensor) was developed to analyze chlorothalonil residues and maximum residue limits (MRLs; 0.5-50 mg/kg) in vegetables in Japan. Conjugates of N-(pentachlorophenoxyacetyl)glycine and bovine serum albumin were covalently coated on the sensor chip. The SPR-sensor quantitatively determined chlorothalonil at concentrations ranging from 8.0 to 44 ng/mL, using TPN9A, a monoclonal antibody to chlorothalonil. The 50% inhibition concentration was 25 ng/mL. The reactivity was 10-fold lower than that of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). However, the SPR-sensor could determine chlorothalonil residues in vegetables at concentrations around the above MRLs. Chlorothalonil spiked in vegetables was recovered at 90-118% within 1 day and at 90-115% across 3 days, correlating with HPLC results. The sensor showed good performance for chlorothalonil residue analysis in vegetables with rapid determination, although the sensitivity and the cross-reactivity were less effective than with the ic-ELISA.
A surface plasmon resonance-based immunosensor (SPR-immunosensor) was developed for the detection of Shiga toxin-producing Escherichia coli (STEC) belonging to the O-antigen groups O26, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The polyclonal antibodies (PoAbs) generated against each of the STEC O-antigen types in rabbits were purified and were immobilized on the sensor chip at 0.5 mg/mL. The limit of detection for STEC O157 by the SPR-immunosensor was found to be 6.3 × 10(4) cells for 75 s. Each of the examined 10 O-antigens on the STECs was detected by the corresponding PoAb with almost no reaction to the other PoAbs. The detected STECs were sufficiently removed from the PoAbs using gelatin or agarose gel without deactivation of the PoAbs, enabling repeatable use of the sensor chip. The developed SPR-immunosensor can be applied for the detection of multiple STEC O-antigens. Furthermore, the new antigen removal technique using the gel displacement approach can be utilized with various immunosensors to improve the detection of pathogens in clinical and public health settings.
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