The specific association of drugs with deoxyoligonucleotides, containing a B-Z junction between left-handed Z-DNA and right-handed B-DNA, was examined by fluorescence and circular dichroism (CD) technique. Ethidium was chosen for a simple DNA binding compound because it binds to right-handed DNA and hybrid B-Z forms containing a B-Z junction in a highly cooperative manner. The binding isotherms were analyzed by an allosteric model in order to describe the cooperativity of association. Binding of ethidium to the DNA that are initially in the hybrid B-Z forms showed over an order of magnitude higher affinity than other DNA which were entirely in the B-form. The conformational transitions of deoxyoligonucleotides containing a B-Z junction as a result of ethidium binding were monitored by CD and the influence of NaCl on the complex formation was also determined by the CD spectra. The singular value decomposition (SVD) analysis was used to characterize a family of CD spectra of the species in binding equilibria. The results of SVD analysis showed a strikingly complex thermodynamic equilibria of cooperative binding of drugs to the allosterically converted DNA forms. The results also showed that these DNA forms in low- and high-salt were different in the absence or presence of drug. These results demonstrate that DNA-binding-drugs can preferentially interact with specific DNA structures and that these interactions are accompanied by allosteric changes of DNA conformations.
Allosteric Regulation/genetics ; Circular Dichroism ; DNA/chemistry* ; Ethidium/chemistry* ; Fluorescent Dyes/chemistry ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides/chemistry ; Sodium Chloride/pharmacology ; Thermodynamics

DNA binding compounds were previously shown to bind to the right-handed DNA forms and hybrid B-Z forms in a highly cooperative manner and indicate that structural specificity plays a key role in a ligand binding to DNA. In this study, the modes of binding and structural specificity of agents to unusual DNA are examined by a variety of fluorescence techniques (intensity, polarization and quenching, etc.) to explore a reliable method to detect the association environment of ligands to deoxyoligonucleotides initially containing a B-Z junction between the left-handed Z-DNA and right-handed B-DNA. The results of fluorescence energy transfer measurement demonstrated that the ligand molecules bind to the allosterically converted DNA structures by intercalation. In the absence of high-resolution structural data, this fluorescence energy transfer measurement allowed reliable measures and infer the binding environment of ligands to the allosteric DNA structures.
Allosteric Regulation ; Circular Dichroism ; DNA/*chemistry/*metabolism ; Energy Transfer ; Ethidium/*metabolism ; Exodeoxyribonucleases/metabolism ; Ligands ; Motion ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides/*metabolism

OBJECTIVETo study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion.

METHODSHepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay.

RESULTSHepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness.

CONCLUSIONHepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.

Apoptosis ; Culture Media ; chemistry ; DNA, Mitochondrial ; genetics ; Ethidium ; chemistry ; Hep G2 Cells ; radiation effects ; Humans ; Radiation Tolerance ; genetics ; Sequence Deletion ; X-Rays

OBJECTIVETo observe the inhibition of flavonoids extract (FE) from Inula britannica on oxidative stress in rat aorta after balloon injury.

METHODThe model of vascular intimal hyperplasia was established by balloon injury. The rats were randomly divided into four groups: control, model, FE and captopril (CAP, positive control). The FE group was treated by intragastric administration with FE in dose of 12.5, 25, 50 mg x kg(-1) x d(-1). The level of malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected by thiobarbituric acid (TAB) method and xanthine oxidase method respectively, and superoxide anion (O2-) in vessel was detected by dihydroethidium (DHE) staining. The histochemistry, immunohistochemistry and Western blot were used to observe the changes of appearance and SOD expression in vascular tissues after balloon injury.

RESULTThe best concentration of FE to rats was 50 mg x kg(-1) x d(-1). The neointima thickness in the model group was significantly higher than that in the FE group (50 mg x kg(-1) x d(-1)) and control group at 14 days after balloon injury (P < 0.01). The lever of MDA in serum of FE group was decreased (P < 0.01) and SOD was increased (P < 0.05) in both serum and vascular tissues. The level of O2*- in the drug group was lower than that in the model group.

CONCLUSIONFE can enhance the antioxidation capacities of vessel tissues by suppressing the formation of O2- induced by injury, by which FE inhibites neointima formation after balloon injury in rat.

Animals ; Antioxidants ; administration & dosage ; chemistry ; Aorta ; drug effects ; metabolism ; Blood Vessels ; drug effects ; metabolism ; Catheterization ; adverse effects ; Down-Regulation ; Ethidium ; analogs & derivatives ; blood ; metabolism ; Flavonoids ; administration & dosage ; chemistry ; Inula ; chemistry ; Male ; Oxidative Stress ; drug effects ; Random Allocation ; Rats ; Superoxide Dismutase ; blood ; metabolism