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International audience; Research on spintronics and on multiferroics leads now to the possibility of combining the properties of these materials in order to develop new functional devices. Here we report the integration of a layer of magnetostrictive material into a magnetic tunnel junction. A FeGa/MgO/Fe heterostructure has been grown on a GaAs(001) substrate by molecular beam epitaxy (MBE) and studied by X-ray magnetic circular dichroism (XMCD). The comparison between magneto optical Kerr effect (MOKE) measurements and hysteresis performed in total electron yield allowed distinguishing the ferromagnetic hysteresis loop of the FeGa top layer from that of the Fe buried layer, evidencing a different switching field of the two layers. This observation indicates an absence of magnetic coupling between the two ferromagnetic layers despite the thickness of the MgO barrier of only 2.5 nm. The in plane magnetic anisotropy has also been investigated. Overall results show the good quality of the heterostructure and the general feasibility of such a device using magnetostrictive materials in magnetic tunnel junction. (C) 2014 Elsevier B.V. All rights reserved

The repair of UV-C (254 nm) DNA lesions by nucleotide excision repair (NER) has been studied in the rodent cell line xrs6 belonging to complementation group 5 of ionising radiation sensitive (IRs) mutants. xrs6 cell line shows a defect in the DNA-end binding protein complex Ku which is involved in the repair of double-strand breaks (DSB) due to IR. In agreement with IR sensitivity, a bleomycin sensitive phenotype of xrs6 cell line was found as compared to the parental CHO-K1 line (factor8 fold). xrs6 exhibited also a slight (factor 2) but reproducible sensitivity to UV-C-light, while a revertant cell line for Ku DNA-end binding activity, xrs6rev, showed a restoration of both IR and UV-C sensitivities to the parental level. The NER activity of these cell lines was measured in vitro in nuclear protein extracts in the presence of plasmid DNA repair substrate damaged with UV-C lesions repaired by NER: xrs6 cell extracts exhibited only 55% of NER activity as compared to the control CHO-K1 and xrs6rev cell extracts. These results indicate that the Ku DSB repair protein is involved also in the NER process.

Nucleotide excision repair (NER) activity was investigated in lymphocytes from patients with chronic lymphocytic leukemia (CLL). The NER process consists of two broad stages: incision/excision of the damaged oligonucleotide and resynthesis of the repair patch. NER in CLL lymphocytes was monitored with the use of in vitro biochemical assays, allowing the determination of either the extent of repair synthesis or the incision activity on damaged plasmid DNA during incubation with whole-cell protein extracts. Fresh CLL tumor cells were purified from the blood of 7 untreated patients and 11 patients who had been treated with chemotherapy. No repair activity was found in 14 extracts (7 treated and 7 untreated) or in normal blood peripheral lymphocytes. The defect was at the level of both repair synthesis and incision/excision activity of DNA damage. In contrast, 4 of the extracts exhibited 25-60% of the repair activity measured in an extract from a control repair-proficient cell line. A linear relationship was found between the values of DNA-repair synthesis and incision activities, which indicates that the extent of significant incision was the limiting factor in these protein extracts. All of the extracts that exhibited DNA-repair activity were purified from lymphocytes of treated patients. These data suggest that chemotherapy might exert an effect on the status of repair activity in the lymphoid tumor cells of patients.

National audience; The cytolethal distending toxin (CDT) is produced by many pathogenic Gram-minus bacteria like Escherichia coli, Helicobacter hepaticus, Haemophilus ducreyi, Salmonella typhi and others. In vivo, the production of CDT by Helicobacter hepaticus induces the development of dysplastic liver nodules, thus defining CDT as a potential carcinogen. Ex vivo, CdtB, the catalytic sub-unit of CDT, is relocated into the eukaryotic cell nucleus. There, CDT induces DNA double-stranded breaks (DSBs), leading to cell cycle arrest and cell death. However, after years of study, the CDT mode of action only begins to be unrevealed and many aspects remain to be studied. To better characterize the CDT-induced DNA lesions, we are studying the biochemistry of CDT, specifically regarding its catalytic nuclease activity and relate these aspects to the overall cellular effects of the toxin. Based on the literature and on the structural homology of CdtB with the DNase I, we developed several CDT mutants for specific residues involved in the catalytic activity. The DNA binding and the nuclease activities of CdtB are studied by in vitro tests, like Supercoiled DNA cleavage and DNA binding assay. Ex vivo, CDT-induced DNA damage and the activation of specific DNA damage response (DDR) pathways have been characterized. Thanks to comet assay and immunofluorescence staining, we have shown that, depending on the CDT dose, DSBs (high doses) or SSBs (moderate doses) will be induced, the later degenerating into DSBs following the replication. In fact, after a treatment with moderate doses, CDT-induced DNA damages cause the activation of the DDR involving the RPA, ATR and CHK1 proteins, characteristic of a replicative stress. The activation of the ATM pathway, due to DSBs induction, occurs later during CDT treatment. The importance of the S-phase passage for the CDT cytotoxicity suggests that proliferating cells are more sensitive to CDT than quiescent cells. The presence of unrepaired damage can lead to cell death, whereas effective repair will allow cells to resume cell cycle. However, improper repair of DNA damage can induce genetic instability and lead to cancer. Bacterial niches containing CDT producing strains are located at epithelia that are quick renewal tissues. Understanding the effects of CDT at the cellular level is an essential step in order to understand these effects at the tissue and/or organism level as well as CDT’s involvement in bacteria pathogenicity.

The 180BR cell line was derived from an acute lymphoblastic leukemia patient who overresponded to radiation therapy and died following radiation morbidity. 180BR cells are hypersensitive to the lethal effects of ionizing radiation and are defective in the repair of DNA double-strand breaks (DSBs). The levels and activity of the proteins of the DNA-dependent protein kinase complex are normal in 180BR cells. To facilitate a measurement of V(D)J recombination, we have characterized 180BRM, a SV40-transformed line derived from 180BR. 180BRM retains the radiosensitivity and defect in DSB repair characteristic of 180BR. The activities associated with DNA-dependent protein kinase are also normal in 180BRM cells. The ability to carry out V(D)J recombination is comparable in 180BRM and a reference control transformed human cell line, MRC5V1. These results show that 180BR and 180BRM differ from the rodent mutants belonging to ionizing radiation complementation groups 4, 5, 6, and 7 and, therefore, represent a new mutant phenotype, in which a defect in DNA DSB rejoining is not associated with defective V(D)J recombination. Furthermore, we have shown that 180BR can arrest at the G1-S and G2-M cell cycle checkpoints after irradiation. These results confirm that 180BR can be distinguished from ataxia telangiectasia.

cis-Dichlorodiammineplatinum(II) (CDDP) resistance in L1210/10 murine leukemia cells is multifactorial and involves decreased drug uptake, increased glutathione content, and enhanced DNA repair activity. We show here that 0.35 M NaCl nuclear extracts from L1210/10 cells possess an approximately 3-fold increase in DNA topoisomerase II activity, compared with parental L1210 cells, as measured by decatenation of kinetoplast DNA. No difference in topoisomerase I activity is observed between the two cell lines. Immunoblot analysis of topoisomerase II protein in resistant and sensitive cells suggests that the observed differences in topoisomerase II activity cannot be explained by differences in the level of protein expressed. L1210/10 cells are 2.5-fold more sensitive than L1210 cells to the cytotoxic effects of the topoisomerase II inhibitor 4'-(9-acridylamino)methane-sulfon-m-anisidide. Sequential treatment with 4'-(9-acridyl-amino)methanesulfon-m-anisidide and CDDP leads to an additive cytotoxic effect of the two drugs in sensitive L1210 cells, as determined by colony formation in semi-solid medium. In contrast, the same treatment leads to a supra-additive effect in L1210/10 cells, which strongly suggests a role for topoisomerase II in the CDDP resistance of this cell line.