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111.
Olga Alster Anna Bielak-Zmijewska Grazyna Mosieniak Maria Moreno-Villanueva Wioleta Dudka-Ruszkowska Aleksandra Wojtala Monika Kusio-Kobia?ka Zbigniew Korwek Alexander Burkle Katarzyna Piwocka Jan K. Siwicki Ewa Sikora 《PloS one》2014,9(8)
Nibrin plays an important role in the DNA damage response (DDR) and DNA repair. DDR is a crucial signaling pathway in apoptosis and senescence. To verify whether truncated nibrin (p70), causing Nijmegen Breakage Syndrome (NBS), is involved in DDR and cell fate upon DNA damage, we used two (S4 and S3R) spontaneously immortalized T cell lines from NBS patients, with the founding mutation and a control cell line (L5). S4 and S3R cells have the same level of p70 nibrin, however p70 from S4 cells was able to form more complexes with ATM and BRCA1. Doxorubicin-induced DDR followed by cell senescence could only be observed in L5 and S4 cells, but not in the S3R ones. Furthermore the S3R cells only underwent cell death, but not senescence after doxorubicin treatment. In contrary to doxorubicin treatment, cells from all three cell lines were able to activate the DDR pathway after being exposed to γ-radiation. Downregulation of nibrin in normal human vascular smooth muscle cells (VSMCs) did not prevent the activation of DDR and induction of senescence. Our results indicate that a substantially reduced level of nibrin or its truncated p70 form is sufficient to induce DNA-damage dependent senescence in VSMCs and S4 cells, respectively. In doxorubicin-treated S3R cells DDR activation was severely impaired, thus preventing the induction of senescence. 相似文献
112.
Andrea Mafficini Eliana Amato Matteo Fassan Michele Simbolo Davide Antonello Caterina Vicentini Maria Scardoni Samantha Bersani Marisa Gottardi Borislav Rusev Giorgio Malpeli Vincenzo Corbo Stefano Barbi Katarzyna O. Sikora Rita T. Lawlor Giampaolo Tortora Aldo Scarpa 《PloS one》2014,9(8)
Background
Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples.Aim
To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity.Methods
35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing.Results
TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis.Conclusions
TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy. 相似文献113.
Human leukemic T lymphocytes (Jurkat cells) were induced to undergo apoptosis by brief irradiation with ultraviolet C light (254 nm). This was accompanied by accumulation of lipid peroxidation products in the form of conjugated dienes, a decrease of total glutathione content, and a shift of its redox state towards the oxidized form. Preincubation of the cells with 1 mM pantothenate resulted in a significant elevation of total glutathione content of the cells, reaching its maximum level, 160% of the control, after 3 h. Similar increase was observed after preincubation with 5 mM N-acetylcysteine, a known precursor of glutathione. Both pantothenic acid and N-acetylcysteine alleviated the ultraviolet-induced decrease of glutathione content, diminished lipid peroxidation, and partly protected the cells against apoptosis produced by ultraviolet irradiation. 相似文献
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116.
This study describes the development of a new colorimetric assay to determine aromatic amino acid aminotransferase (ArAT)
activity. The assay is based on the transamination of l-tryptophan in the presence of 2-oxoglutarate, which yields indole-3-pyruvate (IPyA). The amount of IPyA formed was quantified
by reaction with the Salkowski reagent. Optimized assay conditions are presented for ArAT isozymes isolated from Pseudomonas putida. For comparative purposes, ArAT activity was also determined by high-performance liquid chromatography. ArAT activity staining
in polyacrylamide gels with the Salkowski reagent is also presented. 相似文献
117.
118.
Sikora B Chen Y Lichti CF Harrison MK Jennings TA Tang Y Tackett AJ Jordan JB Sakon J Cameron CE Raney KD 《The Journal of biological chemistry》2008,283(17):11516-11525
HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro. 相似文献
119.
120.
Guber AK Shelton DR Pachepsky YA Sadeghi AM Sikora LJ 《Applied and environmental microbiology》2006,72(12):7531-7539
Modeling release of fecal coliforms is an important component of fate and transport simulations related to environmental water quality. Manure constituents other than fecal coliforms may serve as natural tracers of fecal contamination provided that their release from manure to runoff is similar to the fecal coliform release. The objectives of this work were to compare release of fecal coliforms (FC), chloride (Cl-), organic carbon (OC), and water-soluble phosphorus (P) from dissolving manure and to assess the performance of three models in describing the observed release. Bovine manure was applied on 0.5- by 0.3-m bare and vegetated subplots with 20% slope on sandy loam and clay loam soils. Concentrations of Cl-, FC, OC, and P were measured in runoff collected from troughs at the edges of the subplots at 5-min intervals during 1-h rainfall simulations. The one-parametric exponential model and two-parametric Vadas-Kleinman-Sharpley model and Bradford-Schijven model were fitted to the data. The Bradford-Schijven model had uncorrelated parameters, one of which was linearly related to the irrigation rate, and another parameter reflected the presence or the absence of vegetation. Kinetics of the FC release from manure was similar to the release kinetics of P and OC. The Bradford-Schijven model is recommended to simulate the release of manure constituents. 相似文献