Helicobacter pylori infection has been considered as a risk factor for gastric carcinoma. Strong evidence exists that reactive oxygen species (ROS) play an important role in carcinogenesis, and in vivo investigations have shown increased synthesis of ROS in the gastric mucosa of H.pylori-infected patients. In the present study the direct effects of H.pylori on ROS and DNA synthesis, induction of apoptosis and DNA repair were investigated in the gastric epithelial cell lines AGS and HM02. Incubation of gastric cells with H.pylori extract induced the synthesis of ROS, diminished the levels of reduced glutathione (GSH), induced DNA fragmentation and increased DNA synthesis in gastric cells. Poly(ADP-ribose) formation was increased in gastric cells exposed to H.pylori extract. FACS analysis of gastric cells exposed to H.pylori extract did not reveal any change in the percentage of cells in the G(2)/M phase of the cell cycle. The radical scavengers MnTBAP (a cell permeable superoxide dismutase mimic), ebselen (a GSH peroxidase mimic) and high doses of catalase completely blocked H.pylori extract-induced elevation in DNA synthesis. Our results indicate that H.pylori extract directly induces the synthesis of ROS in gastric epithelial cells and causes DNA damage.
Lethal oxidative stress was investigated in the dinoflagellate Gonyaulax polyedra by measuring the dying-peak of bioluminescence during circadian phases of low physiological light emission, low bioluminescence capacity, and low sensitivity to stimulatory agents. Measurements were carried out in constant darkness after transfer of cells from light at CT 6 (circadian time, 0600 hr). H2O2 (0.08 mM), when administered 1 hr after transfer of cells, led to a multifold, long-lasting enhancement of light emission, which is typical for lethal cell damage. At the circadian phases of investigation, melatonin did not substantially stimulate bioluminescence up to concentrations of 0.5 mM. At this concentration, addition of melatonin prevented the dying-peak and reduced bioluminescence to almost basal values. The high concentration of melatonin applied is not unphysiological in Gonyaulax, because the indoleamine can increase to levels of several millimolar, e.g., in response to temperature signals. These protective effects of melatonin seem to be caused mainly by the direct action of melatonin as an antioxidant, because the major enzymes of antioxidative protection were not stimulated by melatonin, although some of them responded to H2O2. The activities of neither superoxide dismutase, hemoperoxidase/catalase, glutathione peroxidase, nor haloperoxidase were enhanced under the influence of melatonin; glutathione S-transferase activity increased only slightly.
Heat stability was determined by heating the H.pylori cytosolic extract to 95°C for 30 min. For digestion of protein, the bacterial extract was incubated with 0.1 µg/ml trypsin for 2 h at 37°C. The reaction was stopped with soy bean trypsin inhibitor. For preliminary sizing of the protein, the cytosolic
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