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Transient generation of reactive oxygen or nitrogen (ROS/RNS), detected with dihydrodichlorofluoroscein by fluorescence microscopy, occurs within minutes of exposing cells to ionizing radiation. In the 1-10 Gy dose range, the amount of ROS/RNS produced/cell is constant, but the percentage of producing cells increases with dose (20 to 80%). Reversible(More)
The effects of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 on the apoptotic response of U937 monocytic leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) were examined. After a 6-h exposure to 1 microM ara-C, cells stably transfected with a p21WAF1/CIP1 antisense construct were significantly more sensitive to the induction(More)
The rat mitochondrial single strand DNA binding protein (SSB) P16 was purified to apparent homogeneity by elution from single strand DNA agarose with ethidium bromide. Each monomer of P16 contains two tryptophan residues, and the intrinsic fluorescence from these residues is quenched upon binding to single strand polynucleotides. From fluorescence quench(More)
Mitochondrial DNA (mtDNA)-protein complexes were released from the organelles by sodium dodecyl sulfate-lysis and purified by Phenyl-Sepharose CL-4B chromatography. The mitochondrial DNA-binding protein P16 was the only detectable protein in the complex. Treatment of the complex with proteinase K, or subtilisin, revealed the presence of a(More)
This study identified 33 different deletions in mitochondrial DNA from four aging Fischer-344 rat brains and from a cultured rat lymphoma cell line (Nb2 cells). The deletions were located in the longer arc between the heavy and light strand origins of replication. PCR products that spanned across the deleted regions were sequenced, and deletions ranging(More)
The 5'- and 3'-tRNA processing nucleases have been isolated from rat liver mitochondria. The two activities co-purified through heparin-agarose and phenyl-Sepharose columns and then efficiently separated on a DEAE-cellulose column. The 5' processing nuclease was found in the flow-through fraction, and the 3' processing activity eluted with 0.5 M KCl. Both(More)
Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex. Analysis of the(More)
A highly folded, rapidly sedimenting form of rat liver mitochondrial DNA has been released from the organelles wiht BRIJ 58 and sodium deoxycholate in the presence of 0.5 M NaCl and isolated by sedimentation velocity in sucrose gradients. Under these conditions a majority of the mitochondrial DNA labeled in vitro sedimented beyond 39 S, the sedimentation(More)
The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE.(More)