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Hyperoxic stress in normal and mitochondrially-damaged subjects
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L!!!!L! NADPH-OXIDASE ENDOTHELIUM
COMPONENTS
IN THE PULMONARY
Productionof reduced oxygen species(ROS)by pubnonaxy endotheliumhas been implicatedin lung ischemic injury. Previously, we have shown the role of NADPH oxidase in blood-freelung ischemicROS generationand the presence of the cytosolic component p47p” of the enzyme in the rat and mouse lung endotheliumand in bovine pulmonary artery endothelialcells. The goal of the present work was immunohistofluorographiclocalizationof the plasma-membranecomponentsgp91pb and p22 phaand of the cytosolic components@7 p)mand p67 Fhmin the mouseand rat lungsusingmom and polyclonal antibodies. Blood-free lungs from rats and wild type mice and from @l pha-knockoutmice were fixed, freezesectioned, permeabilizedand probed with mouse anti-humangp91ph”/pZ2” monoclonal or rabbit anti-human p4?‘/p67ph” polyclonal antibodies or non-immune IgG. Texas Red-conjugated gvat anti-mouse or anti-rabbit IgG antibody served as the secondary. Epifluorescence digital micrographs showed that gp91@’ and p22 *are present in endothelialcells and smooth muscle cells in the wild-type mouse lung. Absenceof gp91 P”“andpresenceofp22*inthe gP91P”‘-knockoutmouse lung indicatedthe specificityof the monoclonalantibodiesand the efficiencyof the knockout.We also found the expressionof both p47p’“’ and p67”D”in the rat and mouse lung endothelium.The presenceof the componentsof NADPH oxidase makes it a possible source of ROS generation in pulmonary endothelial cells. Supported by Parker B. Francis Fellowship (ABA) and HL-41939 & HL-52565 (ABFI
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THE INSTANT WATER STERILIZING SYSTEM USING ACTIVE OXYGEN SPECIES h&sat&i Azum&l, Mcgumi Hnttmi’2, JunjiOgaam*Z, andNotimichi Kawashima~2. Human R&arch 63 Dewlqmmt, Alma Inc. 1, Toin Unionsily of Yokohm&2 Recently, active oxygen species have attracted much attention in the field of biology, because active oxygen species are
produced in biological systems. They have a strong sterilizing effect and is generated inside a human and animal body to protect it from bacteria. In this study, the novel sterilizing svstemAiECOsvstemAi%v u5ineactiveoxveens~ecieswa.5 &oduced and its sterilkkion effect was t&k&d. In order to generate effectively reactive oxygen, the ECOSYSTEM uses three different wavelengths of UV light The system has its own unique irradiation method which only utilizes the energy light to generate “Hydroxyl-radical” for the complete sterilization of water or otlier llq&da. Needless to say, tke treated water is very safe for the remained small amount of ozone. and active oxygen, generated in the treatment process, &e immediately changed to the orIgina oxygen and water. Sterlking of LEGIONELLA PNEUMOPHILA-ATCC. 33154,0-157. POLO VIRUS TYPE GLCS zab, and BACILLUi STEAROTHERMOPHILUS, IFO-13737 was carried out in active oxygen species genera&l by EC0 system. Furthermore,
disinfection of algae and dye solution waste treatment were also examined.
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MITOCHONDRIA AS A SOURCE OF REAClIVE OXYGEN SPECIES DURING ETHANOL OXIDATION Shannon and Carol C. Cunningham. Wake Forest University School of Medicine, Winston-Salem, NC 27157 The purpose of this study was to evaluate the role of mitcchonchia in generating reactive oxygen species (ROS) elicited during ethanol oxidation. Rat hepatocytes were. incubated with 2’,7’-dichlorofluorescin &rate for 60 min to detect ROS and lactate dehydrogenase leakage was used to assess viability. Incubation with ethanol (1 and 10 mM) stimulated ROS uroduction. increased the cellular NAiX-kNAD’ ratio, and decreased hepatocyk viability slightly, which were reversed by Cmethylpymzole, an inhibitor of alcohol dehydrogenase. Cyanamide, an acctaldehyde dehydrogenase inhibitor, also abolished ethanol-induced ROS production. Ethanol-elicited ROS production was increased severalfold by pretreatment with the mitochondxial complex III inhibitor, antimycin. which markedly increased cell death. The NADH dehydrogenase inhibitor, rotenone, will allow electron flux through FMN, but inhibits transfer to comulex III. Rotenone increased ROS levels compared to untreated and ethanol-treated hepatocytes, but diminished ethanol-induced ROS production in ant&y&treated cells. Diphenyliodonium which inhibits electron flux through FMN, anenuated ethanol-induced ROS production in hepamcytes incubated with or without antimycin. Experiments with mimchondrial inhibitors illustrate that ethanol stimulates ROS production in the NADH dehydrogenase complex, as well as in complex III. These data illustrate that hepatocyte levels of ROS are influenced by the availability of ethanol-generated NADH for
oxidation by the mitochondrial electron transport chain. Support: NIAAA grants 02887 and 5132 07565.
OXYGEN
HYPEROXIC STRESS IN NORMAL AND MlTOCHONDRIALLY-DAMAGED SUBJECTS Britton Chance, William Bank, Hong Long, Department of Biochemistry & Biophysics, University of Pennsylvania Tissue oxygen is stictly regulated at relatively low values over resting intervals which occupy the majority of time in a 24hour cycle. In violent exercise, extreme hypoxia characterizes the tissue oxygen in well trained athletes, reducing tissue oxygen near the level obtained with cuff ischemia (Takafumi and Miwa). At cessation of exercise, however, a massive hyperemia occu& in which the opened vascularity and high flow may cause tissue oxygen to be out of regulation and rise to high levels in which hemoglobin becomes completely saturated. It is estimated that the tissue oxygen level rises to the arterial level. Thus, we can indicate the fraction of the total time that the muscle is hyperoxic and subject to free radical damage as due to one or two minutes Since this occurs following termination of extreme exercise. only at the cessation of exercise, the fraction of the 24 hour interval at which the muscle is exposed to near arterial saturation is estimated to be less than 1% for an athletic individual. Much more significant hyperoxygenation in tissue occurs during the “paradoxical oxygenation” occurring in subjects with rnitochondrial disease, where mild or even the most strenuous exercise of which they are capable causes rises from normal resting level to significant hieher values. estimated to cause saturayions approaihing the &t&al levkl. It would seem therefore that if indeed muscle hyperoxia is to be sought as a source of free radicals in an intervai of exposure to high revels of free radical and possible damage, then indeed the mildly exercised mitochondrially diseased subjects should be intensely investigated.
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L!!!!L! NADPH-OXIDASE ENDOTHELIUM
COMPONENTS
IN THE PULMONARY
Productionof reduced oxygen species(ROS)by pubnonaxy endotheliumhas been implicatedin lung ischemic injury. Previously, we have shown the role of NADPH oxidase in blood-freelung ischemicROS generationand the presence of the cytosolic component p47p” of the enzyme in the rat and mouse lung endotheliumand in bovine pulmonary artery endothelialcells. The goal of the present work was immunohistofluorographiclocalizationof the plasma-membranecomponentsgp91pb and p22 phaand of the cytosolic components@7 p)mand p67 Fhmin the mouseand rat lungsusingmom and polyclonal antibodies. Blood-free lungs from rats and wild type mice and from @l pha-knockoutmice were fixed, freezesectioned, permeabilizedand probed with mouse anti-humangp91ph”/pZ2” monoclonal or rabbit anti-human p4?‘/p67ph” polyclonal antibodies or non-immune IgG. Texas Red-conjugated gvat anti-mouse or anti-rabbit IgG antibody served as the secondary. Epifluorescence digital micrographs showed that gp91@’ and p22 *are present in endothelialcells and smooth muscle cells in the wild-type mouse lung. Absenceof gp91 P”“andpresenceofp22*inthe gP91P”‘-knockoutmouse lung indicatedthe specificityof the monoclonalantibodiesand the efficiencyof the knockout.We also found the expressionof both p47p’“’ and p67”D”in the rat and mouse lung endothelium.The presenceof the componentsof NADPH oxidase makes it a possible source of ROS generation in pulmonary endothelial cells. Supported by Parker B. Francis Fellowship (ABA) and HL-41939 & HL-52565 (ABFI
1 26
25
I
1
THE INSTANT WATER STERILIZING SYSTEM USING ACTIVE OXYGEN SPECIES h&sat&i Azum&l, Mcgumi Hnttmi’2, JunjiOgaam*Z, andNotimichi Kawashima~2. Human R&arch 63 Dewlqmmt, Alma Inc. 1, Toin Unionsily of Yokohm&2 Recently, active oxygen species have attracted much attention in the field of biology, because active oxygen species are
produced in biological systems. They have a strong sterilizing effect and is generated inside a human and animal body to protect it from bacteria. In this study, the novel sterilizing svstemAiECOsvstemAi%v u5ineactiveoxveens~ecieswa.5 &oduced and its sterilkkion effect was t&k&d. In order to generate effectively reactive oxygen, the ECOSYSTEM uses three different wavelengths of UV light The system has its own unique irradiation method which only utilizes the energy light to generate “Hydroxyl-radical” for the complete sterilization of water or otlier llq&da. Needless to say, tke treated water is very safe for the remained small amount of ozone. and active oxygen, generated in the treatment process, &e immediately changed to the orIgina oxygen and water. Sterlking of LEGIONELLA PNEUMOPHILA-ATCC. 33154,0-157. POLO VIRUS TYPE GLCS zab, and BACILLUi STEAROTHERMOPHILUS, IFO-13737 was carried out in active oxygen species genera&l by EC0 system. Furthermore,
disinfection of algae and dye solution waste treatment were also examined.
I 27 I
/
MITOCHONDRIA AS A SOURCE OF REAClIVE OXYGEN SPECIES DURING ETHANOL OXIDATION Shannon and Carol C. Cunningham. Wake Forest University School of Medicine, Winston-Salem, NC 27157 The purpose of this study was to evaluate the role of mitcchonchia in generating reactive oxygen species (ROS) elicited during ethanol oxidation. Rat hepatocytes were. incubated with 2’,7’-dichlorofluorescin &rate for 60 min to detect ROS and lactate dehydrogenase leakage was used to assess viability. Incubation with ethanol (1 and 10 mM) stimulated ROS uroduction. increased the cellular NAiX-kNAD’ ratio, and decreased hepatocyk viability slightly, which were reversed by Cmethylpymzole, an inhibitor of alcohol dehydrogenase. Cyanamide, an acctaldehyde dehydrogenase inhibitor, also abolished ethanol-induced ROS production. Ethanol-elicited ROS production was increased severalfold by pretreatment with the mitochondxial complex III inhibitor, antimycin. which markedly increased cell death. The NADH dehydrogenase inhibitor, rotenone, will allow electron flux through FMN, but inhibits transfer to comulex III. Rotenone increased ROS levels compared to untreated and ethanol-treated hepatocytes, but diminished ethanol-induced ROS production in ant&y&treated cells. Diphenyliodonium which inhibits electron flux through FMN, anenuated ethanol-induced ROS production in hepamcytes incubated with or without antimycin. Experiments with mimchondrial inhibitors illustrate that ethanol stimulates ROS production in the NADH dehydrogenase complex, as well as in complex III. These data illustrate that hepatocyte levels of ROS are influenced by the availability of ethanol-generated NADH for
oxidation by the mitochondrial electron transport chain. Support: NIAAA grants 02887 and 5132 07565.
OXYGEN
HYPEROXIC STRESS IN NORMAL AND MlTOCHONDRIALLY-DAMAGED SUBJECTS Britton Chance, William Bank, Hong Long, Department of Biochemistry & Biophysics, University of Pennsylvania Tissue oxygen is stictly regulated at relatively low values over resting intervals which occupy the majority of time in a 24hour cycle. In violent exercise, extreme hypoxia characterizes the tissue oxygen in well trained athletes, reducing tissue oxygen near the level obtained with cuff ischemia (Takafumi and Miwa). At cessation of exercise, however, a massive hyperemia occu& in which the opened vascularity and high flow may cause tissue oxygen to be out of regulation and rise to high levels in which hemoglobin becomes completely saturated. It is estimated that the tissue oxygen level rises to the arterial level. Thus, we can indicate the fraction of the total time that the muscle is hyperoxic and subject to free radical damage as due to one or two minutes Since this occurs following termination of extreme exercise. only at the cessation of exercise, the fraction of the 24 hour interval at which the muscle is exposed to near arterial saturation is estimated to be less than 1% for an athletic individual. Much more significant hyperoxygenation in tissue occurs during the “paradoxical oxygenation” occurring in subjects with rnitochondrial disease, where mild or even the most strenuous exercise of which they are capable causes rises from normal resting level to significant hieher values. estimated to cause saturayions approaihing the &t&al levkl. It would seem therefore that if indeed muscle hyperoxia is to be sought as a source of free radicals in an intervai of exposure to high revels of free radical and possible damage, then indeed the mildly exercised mitochondrially diseased subjects should be intensely investigated.
’ 9
8
S2I