306 research outputs found

    Mutational Specificity of γ-Radiation-Induced Guanine−Thymine and Thymine−Guanine Intrastrand Cross-Links in Mammalian Cells and Translesion Synthesis Past the Guanine−Thymine Lesion by Human DNA Polymerase η†

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    ABSTRACT: Comparative mutagenesis of γ- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8 % of progeny contained targeted base substitutions, whereas 10.0 % showed semitargeted single-base substitutions. Of the targeted mutations, the G f T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5 ′ and three bases 3 ′ to the cross-link. The most prevalent semitargeted mutation was a C f T transition immediately 5 ′ to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of ∼16%, and both included a dominant G f T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5 ′ to the lesion was increased and 3 ′ to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called “A-rule”. To determin

    Facile formation of highly mobile supported lipid bilayers on surface-quaternized pH-responsive polymer brushes

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    Poly(2-dimethylamino)ethyl methacrylate) (PDMA) brushes are grown from planar substrates via surface atom transfer radical polymerization (ATRP). Quaternization of these brushes is conducted using 1-iodooctadecane in n-hexane, which is a non-solvent for PDMA. Ellipsometry, AFM, and water contact angle measurements show that surface-confined quaternization occurs under these conditions, producing pH-responsive brushes that have a hydrophobic upper surface. Systematic variation of the 1-iodooctadecane concentration and reaction time enables the mean degree of surface quaternization to be optimized. Relatively low degrees of surface quaternization (ca. 10 mol % as judged by XPS) produce brushes that enable the formation of supported lipid bilayers, with the hydrophobic pendent octadecyl groups promoting in situ rupture of lipid vesicles. Control experiments confirm that quaternized PDMA brushes prepared in a good brush solvent (THF) produce non-pH-responsive brushes, presumably because the pendent octadecyl groups form micelle-like physical cross-links throughout the brush layer. Supported lipid bilayers (SLBs) can also be formed on the non-quaternized PDMA precursor brushes, but such structures proved to be unstable to small changes in pH. Thus, surface quaternization of PDMA brushes using 1-iodooctadecane in n-hexane provides the best protocol for the formation of robust SLBs. Fluorescence recovery after photobleaching (FRAP) studies of such SLBs indicate diffusion coefficients (2.8 ± 0.3 μm s–1) and mobile fractions (98 ± 2%) that are comparable to the literature data reported for SLBs prepared directly on planar glass substrates

    Photosynthetic electron flow affects H2O2 signaling by inactivation of catalase in Chlamydomonas reinhardtii

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    A specific signaling role for H2O2 in Chlamydomonas reinhardtii was demonstrated by the definition of a promoter that specifically responded to this ROS. Expression of a nuclear-encoded reporter gene driven by this promoter was shown to depend not only on the level of exogenously added H2O2 but also on light. In the dark, the induction of the reporter gene by H2O2 was much lower than in the light. This lower induction was correlated with an accelerated disappearance of H2O2 from the culture medium in the dark. Due to a light-induced reduction in catalase activity, H2O2 levels in the light remained higher. Photosynthetic electron transport mediated the light-controlled down-regulation of the catalase activity since it was prevented by 3-(3′4′-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. In the presence of light and DCMU, expression of the reporter gene was low while the addition of aminotriazole, a catalase inhibitor, led to a higher induction of the reporter gene by H2O2 in the dark. The role of photosynthetic electron transport and thioredoxin in this regulation was investigated by using mutants deficient in photosynthetic electron flow and by studying the correlation between NADP-malate dehydrogenase and catalase activities. It is proposed that, contrary to expectations, a controlled down-regulation of catalase activity occurs upon a shift of cells from dark to light. This down-regulation apparently is necessary to maintain a certain level of H2O2 required to activate H2O2-dependent signaling pathways

    Immune Cell Recruitment and Cell-Based System for Cancer Therapy

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    Immune cells, such as cytotoxic T lymphocytes, natural killer cells, B cells, and dendritic cells, have a central role in cancer immunotherapy. Conventional studies of cancer immunotherapy have focused mainly on the search for an efficient means to prime/activate tumor-associated antigen-specific immunity. A systematic understanding of the molecular basis of the trafficking and biodistribution of immune cells, however, is important for the development of more efficacious cancer immunotherapies. It is well established that the basis and premise of immunotherapy is the accumulation of effective immune cells in tumor tissues. Therefore, it is crucial to control the distribution of immune cells to optimize cancer immunotherapy. Recent characterization of various chemokines and chemokine receptors in the immune system has increased our knowledge of the regulatory mechanisms of the immune response and tolerance based on immune cell localization. Here, we review the immune cell recruitment and cell-based systems that can potentially control the systemic pharmacokinetics of immune cells and, in particular, focus on cell migrating molecules, i.e., chemokines, and their receptors, and their use in cancer immunotherapy

    Chemistry and Biology of DNA Containing 1,N2-Deoxyguanosine Adducts of the α,β-Unsaturated Aldehydes Acrolein, Crotonaldehyde, and 4-Hydroxynonenal

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