aDepartment of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, Chiba 274-8510, Japan
bAging Regulation, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
cKajiyama Clinic, Kyoto 615-0035, Japan
dResearch Team for Molecular Biomarker, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
eDepartment of Endocrinology and Metabolism, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto 602-8566, Japan
fI’rom Pharmaceutical Co., LTD., Tokyo 141-0032, Japan
gDepartment of Medical Biochemistry, Kobe Pharmaceutical University, Kobe 658-8566, Japan
hInstitute of Bio-Response Informatics, Kyoto 612-8016, Japan
Received 31 July 2008.
Available online 14 August 2008.
Abstract
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.
Keywords: Ascorbic acid; Chemiluminescence; Gluconolactonase; Hydrogen-rich pure water; Oxidative stress; ROS; Senescence marker protein-30; Superoxide; Vitamin C
Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan. ishigami@tmig.or.jp
Abstract
Senescence marker protein-30 (SMP30) is a calcium-binding protein that decreases in an androgen-independent manner with aging. To elucidate the physiological role of this protein, we introduced a null mutation of the SMP30 gene into the germ line of mice. Despite the complete lack of SMP30 (SMP30-/-), these mutant mice were indistinguishable from their wild-type (SMP30+/+) littermates in terms of development and fertilization capability. We then investigated the tissue susceptibility for apoptosis induced by cytokine using primary cultured hepatocytes, because SMP30 could rescue cells from cell death caused by calcium influx, using a calcium ionophore as previously described. SMP30-/- hepatocytes were found to be more susceptible to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plus actinomycin D (ActD) than SMP30+/+ hepatocytes. In addition, the TNF-alpha/ActD-induced caspase-8 activity in SMP30-/- hepatocytes was twofold greater than that in SMP30+/+ hepatocytes. In contrast, no significant difference was observed in the TNF-alpha/ActD-induced nuclear factor-kappa B activation of SMP30+/+ versus SMP30-/- hepatocytes, indicating that SMP30 is not related to TNF-alpha/ActD-induced nuclear factor-kappa B activation itself. Moreover, deletion of the SMP30 gene enhanced liver injury after treatment in vivo with anti-Fas antibody and the SMP30+/- mice showed intermediate susceptibility to Fas-induced apoptosis. Collectively, these results demonstrate that SMP30 acts to protect cells from apoptosis.
PMID: 12368201 [PubMed - indexed for MEDLINE]PMCID: PMC1867294Free PMC Article
Targeted disruption of the SMP30 gene. A: Partial restriction maps of the wild-type SMP30 locus, targeting vector, and mutant locus. To generate the mutated locus, exon III was disrupted by insertion of a positive selection marker (neo) and dual-negative selection markers (tk and Pr/DT-A). The sense and an...
Intracellular localization of SMP30. Primary cultured hepatocytes from SMP30+/+ ( A, B), SMP30+/− (C, D), and SMP30−/− (E, F) mice were stained with rabbit anti-mouse SMP30 and FITC-labeled goat anti-rabbit IgG (A, C, E). Actin fibers were detected by using rhodamine-phalloidin (B, D, F). Fluorescence was image...
Induction of apoptosis by TNF-α/ActD. SMP30+/+ ( A, C, E) and SMP30−/− (B, D, F) hepatocytes were incubated for 24 hours in medium lacking (A, B) or containing 20 ng/ml of TNF-α plus 10 ng/ml of ActD (C, D) or 100 ng/ml of ActD (E, F). Apoptosis was detected by staining with both FITC-conjugated annexin V (ligh...
TNF-α/ActD-induced cell death. SMP30+/+ and SMP30−/− hepatocytes were incubated for 24 hours in medium containing 20 ng/ml of TNF-α plus 1 ng/ml, 10 ng/ml, or 100 ng/ml of ActD. Percentage of survival was calculated relative to untreated controls by XTT assay. Values are expressed as average percent...
Induction of caspase-8 enzyme activity by TNF-α/ActD treatment. Apoptosis was induced in SMP30+/+ and SMP30−/− hepatocytes by treatment with 20 ng/ml of TNF-α plus 10 ng/ml of ActD for 10 hours. Data are expressed as percent activity of caspase-8 enzyme relative to basal levels (ie, pretreatment = 1...
TNF-α/ActD-induced NF-κB activation. SMP30+/+ and SMP30−/− hepatocytes were treated with 20 ng/ml of TNF-α and 10 ng/ml of ActD for 60 minutes. Nuclear extracts were prepared as described in Materials and Methods and used in an EMSA to measure NF-κB DNA-binding activity using a NF-κB-specific oligo-...
Anti-Fas antibody treatment in vivo. SMP30+/+, SMP30+/−, and SMP30−/− mice were injected via tail vein with anti-Fas antibody (3 μg/25 g mouse body weight). Mice were sacrificed after a 6-hour treatment with anti-Fas antibody. A: Serum ALT levels were measured. ALT values in control samples were less than 40...
