Hydrogen-rich pure water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/GNL knockout mice

Hydrogen is an established anti-oxidant that prevents acute oxidative stress. To clarify the mechanism of hydrogen’s effect in the brain, we administered hydrogen-rich pure water (H₂) to senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), also a well-known anti-oxidant. These KO mice were divided into three groups; recipients of H₂, VC, or pure water (H₂O), administered for 33 days. VC levels in H₂ and H₂O groups were <6% of those in the VC group. Subsequently, superoxide formation during hypoxia-reoxygenation treatment of brain slices from these groups was estimated by a real-time biography imaging system, which models living brain tissues, with Lucigenin used as chemiluminescence probe for superoxide. A significant 27.2% less superoxide formed in the H₂ group subjected to ischemia-reperfusion than in the H₂O group. Thus hydrogen-rich pure water acts as an anti-oxidant in the brain slices and prevents superoxide formation.     

Effect of vitamin C depletion on age-related hearing loss in SMP30/GNL knockout mice

Using senescence marker protein 30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), we examined whether modulating VC level affects age-related hearing loss (AHL). KO and wild-type (WT) C57BL/6 mice were given water containing 1.5 g/L VC [VC(+)] or 37.5 mg/L VC [VC(−)]. At 10 months of age, KO VC(−) mice showed significant reduction in VC level in the inner ear, plasma, and liver, increase in auditory brainstem response (ABR) thresholds, and decrease in the number of spiral ganglion cells compared to WT VC(−), WT VC(+), and KO VC(+) mice. There were no differences in VC level in the inner ear, ABR thresholds, or the number of spiral ganglion cells among WT VC(−), WT VC(+), and KO VC(+) mice. These findings suggest that VC depletion can accelerate AHL but that supplementing VC may not increase VC level in the inner ear or slow AHL in mice.     

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.     

Effect of periplasmic expression of recombinant mouse interleukin-4 on hydrogen peroxide concentration and catalase activity in Escherichia coli

Oxidative stress occurs as a result of imbalance between generation and detoxification of reactive oxygen species (ROS). This kind of stress was rarely discussed in connection with foreign protein production in Escherichia coli. Relation between cytoplasmic recombinant protein expression with H₂O₂ concentration and catalase activity variation was already reported. The periplasmic space of E. coli has different oxidative environment in relative to cytoplasm and there are some benefits in periplasmic expression of recombinant proteins. In this study, hydrogen peroxide concentration and catalase activity following periplasmic expression of mouse IL-4 were measured in E. coli. After construction of pET2mIL4 plasmid, the expression of recombinant mouse interleukin-4 (mIL-4) was confirmed. Then, the H₂O₂ concentration and catalase activity variation in the cells were studied in exponential and stationary phases at various ODs and were compared to those of wild type cells and empty vector transformed cells. It was revealed that empty vector introduction and periplasmic recombinant protein expression increased significantly the H₂O₂ concentration of the cells. However, the H₂O₂ concentration in mIL-4 expressing cells was significantly higher than its concentration in empty vector transformed cells, demonstrating more effects of recombinant mIL-4 expression on H₂O₂ elevation. Likewise, although catalase activity was reduced in foreign DNA introduced cells, it was more lowered following expression of recombinant proteins. Correlation between H₂O₂ concentration elevation and catalase activity reduction with cell growth depletion is also demonstrated. It was also found that recombinant protein expression results in cell size increase.     

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc1: Implications for the mechanism of superoxide production

In addition to its bioenergetic function of building up proton motive force, cytochrome bc₁ can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ_o) formed at the quinone oxidation/reduction Q_o site (Q_o) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b_L (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc₁ that severely impeded the oxidation of FeS by cytochrome c₁, changed density of FeS around Q_o by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Q_o and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQ_o that present a risk of generation of superoxide by cytochrome bc₁. Isolation of this reaction sequence from multiplicity of possible reactions at Q_o helps to better understand conditions under which complex III might contribute to ROS generation in vivo.