19 research outputs found
Origin of pyrimidine deoxyribonucleotide pools in perfused rat heart: implications for 3â˛-azido-3â˛-deoxythymidine-dependent cardiotoxicity
Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides
Commonly used metabolic labels for DNA, including 5-ethynyl-2â˛-deoxyuridine (EdU) and BrdU, are toxic antimetabolites that cause DNA instability, necrosis, and cell-cycle arrest. In addition to perturbing biological function, these properties can prevent metabolic labeling studies where subsequent tissue survival is needed. To bypass the metabolic pathways responsible for toxicity, while maintaining the ability to be metabolically incorporated into DNA, we synthesized and evaluated a small family of arabinofuranosyl-ethynyluracil derivatives. Among these, (2â˛S)-2â˛-deoxy-2â˛-fluoro-5-ethynyluridine (F-ara-EdU) exhibited selective DNA labeling, yet had a minimal impact on genome function in diverse tissue types. Metabolic incorporation of F-ara-EdU into DNA was readily detectable using copper(I)-catalyzed azideâalkyne âclickâ reactions with fluorescent azides. F-ara-EdU is less toxic than both BrdU and EdU, and it can be detected with greater sensitivity in experiments where long-term cell survival and/or deep-tissue imaging are desired. In contrast to previously reported 2â˛-arabino modified nucleosides and EdU, F-ara-EdU causes little or no cellular arrest or DNA synthesis inhibition. F-ara-EdU is therefore ideally suited for pulse-chase experiments aimed at âbirth datingâ DNA in vivo. As a demonstration, Zebrafish embryos were microinjected with F-ara-EdU at the one-cell stage and chased by BrdU at 10 h after fertilization. Following 3 d of development, complex patterns of quiescent/senescent cells containing only F-ara-EdU were observed in larvae along the dorsal side of the notochord and epithelia. Arabinosyl nucleoside derivatives therefore provide unique and effective means to introduce bioorthogonal functional groups into DNA for diverse applications in basic research, biotechnology, and drug discovery
Expression, purification, crystallization and preliminary X-ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine
A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of the VchUPhâthymidine complex (of dimensions âź200â350â
Âľm) were grown by the sitting-drop vapour-diffusion method in âź7â
d at 291â
K. The crystallization solution consisted of 1.5â
Âľl VchUPh (15â
mgâ
ml(â1)), 1â
Âľl 0.1â
M thymidine and 1.5â
Âľl reservoir solution [15%(w/v) PEG 4000, 0.2â
M MgCl(2).6H(2)O in 0.1â
M TrisâHCl pH 8.5]. The crystals diffracted to 2.12â
Ă
resolution and belonged to space group P2(1) (No. 4), with unit-cell parameters a = 91.80, b = 95.91, c = 91.89â
Ă
, β = 119.96°. The Matthews coefficient was calculated as 2.18â
Ă
(3)â
Da(â1); the corresponding solvent content was 43.74%
The X-ray structure of Salmonella typhimurium uridine nucleoside phosphorylase complexed with 2,2'-anhydrouridine, phosphate and potassium ions at 1.86 A resolution.
Uridine nucleoside phosphorylase is an important drug target for the development of anti-infective and antitumour agents. The X-ray crystal structure of Salmonella typhimurium uridine nucleoside phosphorylase (StUPh) complexed with its inhibitor 2,2'-anhydrouridine, phosphate and potassium ions has been solved and refined at 1.86 A resolution (R(cryst) = 17.6%, R(free) = 20.6%). The complex of human uridine phosphorylase I (HUPhI) with 2,2'-anhydrouridine was modelled using a computational approach. The model allowed the identification of atomic groups in 2,2'-anhydrouridine that might improve the interaction of future inhibitors with StUPh and HUPhI