Oxygen sensing was investigated in rat pheochromocytoma PC12 cells. They respond to hypoxia with an increased intracellular generation of reactive oxygen species (ROS), measured by oxidation of dihydrorhodamine 123. This increase is abolished by intracellular superoxide scavenging by Mn(III)-tetrakis(1-methyl-4-pyridyl)-porphyrin, and reduced or absent in the presence of the flavoprotein/complex I inhibitors, diphenyleneiodonium and rotenone. The same inhibitors, but neither intra-nor extracellular (superoxide dismutase) superoxide scavenging, abolish the hypoxia-induced increase in tyrosine hydroxylase (TH) gene expression. Thus, ROS production increases in PC12 cells during hypoxia, but this is not the cause of hypoxic TH mRNA upregulation that involves a flavoprotein.z 1999 Federation of European Biochemical Societies.

An NADPH oxidase complex composed of a membrane-bound flavocytochrome b 558 consisting of two subunits (p22 phox and gp91 phox ) and cytosolic activating factors (p47 phox and p67 phox ) generates superoxide anions from oxygen in the respiratory burst of phagocytic cells. Inconsistent results have been previously obtained concerning its additional occurrence in pulmonary artery endothelial cells (PAEC), and this issue was addressed in the present study. PAEC isolated from porcine pulmonary trunk contained mRNA for p22 phox and gp91 phox as demonstrated by reverse transcription-polymerase chain reaction. Immunohistochemistry demonstrated cytochrome subunits, p22 phox , gp91 phox , p47 phox , and p67 phox , both in vitro in isolated PAEC and in situ in endothelial cells in tissue sections of the pulmonary trunk. Isolated PAEC generated reactive oxygen species (ROS; measured by lucigenin-induced chemiluminescence and conversion of dihydrorhodamine 123 into rhodamine 123) in response to stimulation with phorbol 12-myristate 13-acetate. This stimulated ROS production was sensitive to the flavoprotein inhibitor diphenyleneiodonium, and reduced when the superoxide scavenger superoxide dismutase was added. Chemiluminescence measurements of superoxide generation by stimulated PAEC accounted for approximately 1% of that generated by stimulated peritoneal macrophages. The data demonstrate a low-output NADPH oxidase system in porcine PAEC sharing several components with that identified in phagocytic cells.

The methylation and amplification pattern of genomic DNA of carrot root expiants (Daucus carota L.) undergoes transitory changes during the cultural cycle. A high degree of variation was observed as early as 36 h after the incubation of fresh expiants in the nutrient medium and, depending on the hormonal treatment significant modifications occurred during 14 days of culture. Proliferative tissue conditioned by kinetin showed an extensive reduction in DNA methylation. Changes in the DNA amplification pattern were not necessarily linked to methylation.

No abstract

No abstract