The Experts below are selected from a list of 54 Experts worldwide ranked by ideXlab platform
A. Skladanowski - One of the best experts on this subject based on the ideXlab platform.
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Modulation of G_2 arrest enhances cell death induced by the antitumor 1-nitroacridine derivative, Nitracrine
Apoptosis, 2002Co-Authors: A. SkladanowskiAbstract:Nitracrine (Ledakrin) is an antitumor drug which is activated by cellular enzymes and binds covalently to DNA. Previous studies have shown that covalent binding and Crosslinking of DNA is associated with the cytotoxic and antitumor activities of this compound. In this study, cell cycle perturbations, effects on DNA synthesis and the cell death process initiated by Nitracrine were studied in murine leukemia L1210 cells. We show that exposure of L1210 cells to Nitracrine at the IC_99 concentration delayed progression through the S phase and transiently arrested cells in G_2/M as found by flow cytometry. Higher drug concentration (2 × IC_99) inhibited cell cycle progression in the S phase and induced rapid cell death. Both studied concentrations of the drug produced different effects on DNA synthesis as determined by bromodeoxyuridine incorporation, with a delay in the S phase progression at EC_99 concentration and irreversible arrest in early S phase at the higher dose (2 × IC_99). At both concentrations of Nitracrine cell death occurred preferentially in the S phase as revealed by the TUNEL assay. When cells treated with the drug for 4 hours were post-incubated in the presence of 1 mM caffeine this led to rapid cell death and suppression of the G_2 arrest. This was associated with a about 10-fold increase in the cytotoxicity of Nitracrine. Similar effects were observed for another DNA Crosslinking agent, cis-platinum, and to a lesser extent, for DNA topoisomerase I inhibitor, camptothecin. Together, our studies show that suppression of G_2 arrest induced by Nitracrine greatly enhances its cytotoxicity toward L1210 cells.
Janusz M Gebicki - One of the best experts on this subject based on the ideXlab platform.
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Crosslinking of DNA and proteins induced by protein hydroperoxides
Biochemical Journal, 1999Co-Authors: Silvia Gebicki, Janusz M GebickiAbstract:Exposure of DNA to several proteins peroxidized by radiation-generated hydroxyl free radicals resulted in formation of crosslinks between the macromolecules, detected by retardation and broadening of DNA bands in agarose gels. This technique proved suitable for the study of Crosslinking of DNA with peroxidized BSA, insulin, apotransferrin and alpha casein, but not with several other proteins, including histones. The Crosslinking depended on the presence of intact hydroperoxide groups on the protein, on their number, and on the duration of the interaction with DNA. All DNA samples tested, pBR322, pGEM, lambda/HindIII and pUC18, formed crosslinks with the peroxidized BSA. Sodium chloride and formate prevented the Crosslinking if present during incubation of the peroxidized protein and DNA, but had no effect once the crosslinks had formed. The gel shift of the crosslinked DNA was reversed by proteolysis, indicating that the DNA mobility change was due to attachment of protein and that the Crosslinking did not induce DNA strand breaks. The metal chelators Desferal and neocuproine reduced the extent of the Crosslinking, but did not prevent it. Scavengers of free radicals did not inhibit the crosslink formation. The DNA-protein complex was not disrupted by vigorous agitation, by filtration or by non-ionic detergents. These observations show that the Crosslinking of DNA with proteins mediated by protein hydroperoxides is spontaneous and probably covalent, and that it may be assisted by transition metals. It is suggested that formation of such crosslinks in living organisms could account for some of the well-documented forms of biological damage induced by reactive oxygen species-induced oxidative stress.
Bohdan Skalski - One of the best experts on this subject based on the ideXlab platform.
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highly efficient fluorescent interstrand photo Crosslinking of DNA duplexes labeled with 5 fluoro 4 thio 2 o methyluridine
ChemBioChem, 2014Co-Authors: Joanna Nowakkarnowska, Ziad Chebib, Jan Milecki, Stefan Franzen, Bohdan SkalskiAbstract:The formation of a fluorescent photoadduct between 5-fluoro-4-thiouridine ((FS) U), in the sequence context 5'-A(FS) UA-3' and incorporated into a synthetic oligonucleotide either at its 3'- or 5'-end, and one of the thymines of the TAT motif in a complementary target DNA strand led to photo-Crosslinking of the two strands for several oligonucleotide constructs. Enzymatic digestion, MS, UV, and fluorescence spectral analyses of the interstrand crosslinked oligonucleotides revealed the identity of the thymidine that participates in the photo-Crosslinking reaction as well as the diastereomeric structures of the crosslinks. The proposed pathways of interstrand photo-Crosslinking are supported by experiments with isotopically labeled oligonucleotide constructs and visualized by means of molecular dynamics simulations.
Casey J Krusemark - One of the best experts on this subject based on the ideXlab platform.
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Crosslinking of DNA linked ligands to target proteins for enrichment from DNA encoded libraries
MedChemComm, 2016Co-Authors: Kyle E Denton, Casey J KrusemarkAbstract:Achieving sufficient enrichment of ligands from DNA-encoded libraries for detection can be difficult, particularly for low affinity ligands within highly complex libraries. To address this challenge, we present an approach for Crosslinking DNA-linked ligands to target proteins using electrophilic or photoreactive groups. The approach involves the tethering of a ssDNA oligonucleotide to a DNA-encoded molecule to enable attachment of a reactive group post-synthetically via DNA hybridization. Crosslinking traps ligand–protein complexes while in solution and allows for stringent washing conditions to be applied in the subsequent purification. Five reactive groups (tosyl, NHS ester, sulfonyl fluoride, phenyl azide, and diazirine) were tested for Crosslinking efficiency and specificity with three DNA-linked ligands to their target proteins. In a model selection, Crosslinking resulted in improved enrichment of both high and a low affinity ligands in comparison to a selection with a solid-phase immobilized protein.
Peter Karran - One of the best experts on this subject based on the ideXlab platform.
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Crosslinking of DNA repair and replication proteins to DNA in cells treated with 6 thioguanine and uva
Nucleic Acids Research, 2011Co-Authors: Quentin Gueranger, Azadeh Kia, David Frith, Peter KarranAbstract:The DNA of patients taking immunosuppressive and anti-inflammatory thiopurines contains 6-thioguanine (6-TG) and their skin is hypersensitive to ultraviolet A (UVA) radiation. DNA 6-TG absorbs UVA and generates reactive oxygen species that damage DNA and proteins. Here, we show that the DNA damage includes covalent DNA-protein crosslinks. An oligonucleotide containing a single 6-TG is photochemically crosslinked to cysteine-containing oligopeptides by low doses of UVA. Crosslinking is significantly more efficient if guanine sulphonate (G(SO3))--an oxidized 6-TG and a previously identified UVA photoproduct--replaces 6-TG, suggesting that G(SO3) is an important reaction intermediate. Crosslinking occurs via oligopeptide sulphydryl and free amino groups. The oligonucleotide-oligopeptide adducts are heat stable but are partially reversed by reducing treatments. UVA irradiation of human cells containing DNA 6-TG induces extensive heat- and reducing agent-resistant covalent DNA-protein crosslinks and diminishes the recovery of some DNA repair and replication proteins from nuclear extracts. DNA-protein crosslinked material has an altered buoyant density and can be purified by banding in cesium chloride (CsCl) gradients. PCNA, the MSH2 mismatch repair protein and the XPA nucleotide excision repair (NER) factor are among the proteins detectable in the DNA-crosslinked material. These findings suggest that the 6-TG/UVA combination might compromise DNA repair by sequestering essential proteins.