Naoki Maruyama

Tokyo Metropolitan Institute of Gerontology, Edo, Tōkyō, Japan

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Publications (168)580.15 Total impact

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    ABSTRACT: Recent studies on senescence marker protein-30 (SMP30) have shown that it has an important functional role in the aging process, but its precise participation in cellular works has not been fully determined. We hypothesize that SMP30 plays crucial roles in signaling processes by modulating the balance of protein tyrosine kinase (PTK)/protein tyrosine phosphatase (PTP) and in activating proinflammatory NF-κB. An experimental paradigm of gain and loss of SMP30 function was established using SMP30-overexpressed YPEN-1 cells (herein referred to as "SMP30(+) cells") and SMP30 (Y/-) knockout mouse kidneys. The resulting data show that SMP30 expression suppressed oxidative stress-induced PTK/PTP dysregulation and PP1/2A inactivation in SMP30(+) cells, leading to the suppression of NF-κB activation. In the kidneys of SMP30 (Y/-) mice, SMP30 deficiency was found to induce NF-κB activation via the upstream signaling of NIK/IKK and MAPKs and to upregulate downstream NF-κB-responsive gene expression. In this study, we also demonstrate for the first time that SMP30 deficiency induced PTK activity in SMP30 (Y/-) kidneys, thereby significantly increasing the tyrosine phosphorylation of a catalytic subunit of PP2A (PP2Ac-Tyr307). Based on these findings, we propose that SMP30 involves NF-κB regulation through the PTK/PTP balance and that the age-related decrease of SMP30 causes NF-κB activation, which contributes to an exacerbation of the inflammatory process during aging.
    Journal of Molecular Medicine 11/2014; · 4.74 Impact Factor
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    ABSTRACT: AimSenescence marker protein-30 (SMP30)/gluconolactonase (GNL) is an age-associated protein in that its presence decreases with aging. Here, we used immunohistochemical analysis to investigate the changes of SMP30/GNL in individual cells of the liver from progressively aged mice.Methods Male C57BL/6 strain mice at 1, 3, 6, 12, 24 and 30 months-of-age were the source of hepatic cells used to detect SMP30/GNL. Liver sections from these mice were subjected to immunohistochemical staining with anti-SMP30/GNL antibody. For immunofluorescent staining, primary cultured hepatocytes from mice at various ages were stained with SMP30/GNL and albumin.ResultsIn liver cells from mice of all ages, SMP30/GNL staining appeared in some but not all parenchymal cells, and localized in both the nuclei and cytoplasm. Moreover, SMP30/GNL-positive staining of parenchymal cells was present only around central vein areas, but not at sites of portal veins. Furthermore, the number of SMP30/GNL-positive cells increased as mice aged from 1 to 12 months, then decreased from the 12th to 24th month. Results were similar in primary cultured hepatocytes from mice of various ages.ConclusionsSMP30/GNL-positive cells localized mainly around the central veins in the livers of mice and decreased numerically with aging, although there was no age-related change in counts of albumin-positive cells. SMP30/GNL protein occupied the nuclei and cytoplasm. Therefore, nuclear SMP30/GNL protein might be a regulatory factor specific for genes whose expression governs transcription and the aging process. Geriatr Gerontol Int 2014; ●●: ●●–●●.
    Geriatrics & Gerontology International 11/2014; · 1.58 Impact Factor
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    ABSTRACT: Potato chips (PC) contain abundant amounts of the free-radical scavenger ascorbic acid (AA) due to the rapid dehydration of potato tubers (Solanum tuberosum) that occurs during frying. To evaluate the antioxidant activity of PC, we examined reactive oxygen species (ROS) levels in tissues from SMP30/GNL knockout (KO) mice that cannot synthesize AA and determined AA and ROS levels after the animals were fed 20 and 10% PC diets for 7 weeks. Compared with AA-sufficient mice, AA-depleted SMP30/GNL KO mice showed high ROS levels in tissues. SMP30/GNL KO mice fed a PC diet showed high AA and low ROS levels in brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, eyeball, and epididymal fat compared with AA-depleted mice. Our data suggest that PC intake increases AA levels and enhances ROS scavenging activity in tissues of SMP30/GNL-KO mice, which are a promising model for evaluating the antioxidant activity of foods.
    Journal of Agricultural and Food Chemistry 09/2014; · 3.11 Impact Factor
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    ABSTRACT: Aims/IntroductionSenescence marker protein-30 (SMP30) is abundantly expressed in renal proximal tubule cells, but its expression decreases with age. Previous studies have shown that reduced SMP30 expression could contribute to aging-associated deterioration of cellular function and tissue injury. In the present study, we investigated the effects of SMP30 deficiency on the pathogenesis of diabetic nephropathy.Materials and Methods Diabetes was induced using streptozotocin in male SMP30 knockout mice (KO) and wild-type mice at 7 weeks-of-age. Vitamin C was added to the drinking water to prevent vitamin C deficiency in KO mice. The mice were killed 12 weeks after the induction of diabetes.ResultsUrinary biomarkers for proximal tubule damage were significantly increased in non-diabetic KO mice compared with wild-type mice. Furthermore, diabetes-induced tubular damage was significantly exacerbated by SMP30 deletion. Morphological analysis showed a link between cortical tubulointerstitial fibrosis area and the degree of tubular damage. However, SMP30 deletion did not affect mesangial expansion. Tubular injury was associated with accumulation of hypoxia-inducible factor-1α and increased hypoxia-inducible factor-1α targeted gene expression. SMP30 deletion initiated oxidative stress; however, it did not exacerbate the oxidative stress seen in diabetic mice. In contrast, tubular inflammation was associated with SMP30 deletion only in diabetic mice.Conclusions Based on this evidence, we concluded that SMP30 deficiency exacerbates proximal tubule injury in diabetic mice. Decreased SMP30 could contribute to the increased incidence of various chronic kidney diseases, including diabetic nephropathy, with age.
    Journal of Diabetes Investigation. 07/2014; 6(1).
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    ABSTRACT: We sought to elucidate the effect of an ascorbic acid (AA) deficiency on gene expression, because the water soluble antioxidant AA is an important bioactive substance in vivo. We performed microarray analyses of the transcriptome in the liver from senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which are unable to synthesize AA in vivo. Our microarray analysis revealed that the AA deficiency increased gene expression related to the oxidation-reduction process, i.e., the nuclear factor, erythroid derived 2, like 2 (Nrf2) gene, which is a reactive oxygen species-sensitive transcriptional factor. Moreover, this AA deficiency increased the expression of genes for lipid metabolism including the cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1), which is a late-limiting enzyme of the primary bile acid biosynthesis pathway. Although an AA deficiency increased the Cyp7a1 protein level, bile acid levels in the liver and gallbladder decreased. Since Cyp7a1 has a heme iron at the active site, AA must function as a reductant of the iron required for the continuous activation of Cyp7a1. This experimental evidence strongly supports a role for AA in the physiologic oxidation-reduction process and lipid metabolism including bile acid biosynthesis. General significance Although many effects of AA supplementation have been reported, no microarray analysis of AA deficiency in vivo is available. Results from using this unique model of AA deficiency, the SMP30/GNL-KO mouse, now provide new information about formerly unknown AA functions that will implement further study of AA in vivo.
    Biochimica et Biophysica Acta 04/2014; · 4.66 Impact Factor
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    ABSTRACT: Background The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse, a model of human crescentic glomerulonephritis (CrGN) and systemic vasculitis, is characterized by the production of MPO-ANCA and marked leukocytosis. This study was performed to identify the specific populations of leukocytes associated with CrGN and susceptibility loci for pathogenic leukocytosis.Methods420 female (C57BL/6 × SCG/Kj) F2 intercross mice were subjected to serial flow cytometry examination of the peripheral blood (PB). Kidney granulocytes and monocytes were histopathologically examined. Linkage analyses were done with 109 polymorphic microsatellite markers.ResultsCorrelation studies revealed that increase of the granulocytes, F4/80+ cells, CD3+CD4-CD8- T cells, and dendritic cells (DCs) in PB were significantly associated with glomerulonephritis, crescent formation and vasculitis. In kidney sections, F4/80low cells were observed in crescent, while F4/80high cells were around the Bowman's capsules and in the interstitium. Numbers of F4/80+ cells in crescents significantly correlated with F4/80+ cell numbers in PB, but not with numbers of F4/80+ cells in the interstitium. Genome-wide quantitative trait locus (QTL) mapping revealed three SCG/Kj-derived non-Fas QTLs for leukocytosis, two on chromosome 1 and one on chromosome 17. QTLs on chromosome 1 affected DCs, granulocytes and F4/80+ cells, but QTL on chromosome 17 affected DCs and granulocytes.Conclusion We found CrGN-associated leukocytes and susceptibility QTLs with their positional candidate genes. F4/80+ cells in crescents are considered as recruited inflammatory macrophages. The results provide information for leukocytes to be targeted and genetic elements in CrGN and vasculitis.
    Clinical & Experimental Immunology 03/2014; · 3.28 Impact Factor
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    ABSTRACT: Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30) is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA) biosynthesis. SMP30 also participates in Ca2+ efflux by activating the calmodulin-dependent Ca2+-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO) mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15–24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1) higher plasma levels of triglyceride and aspartate aminotransferase; (2) severe accumulation of hepatic triglyceride and total cholesterol; (3) higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4) decreased mRNA and protein levels of Apolipoprotein B (ApoB) in livers – ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion.
    FEBS Open Bio. 01/2014;
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    ABSTRACT: Individuals born with a low birth weight (LBW) have a higher risk of developing kidney dysfunction during their lifetime and sometimes exhibit focal segmental glomerulosclerosis (FSGS) lesions in their glomeruli. We herein try to obtain other pathological characteristics of LBW-related nephropathy.
    Diagnostic Pathology 01/2014; 9(1):181. · 2.41 Impact Factor
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    ABSTRACT: Senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice are incapable of synthesizing L-ascorbic acid (AA) in vivo. As AA is known to be a water-soluble anti-oxidant, we assessed protein oxidation levels in livers from SMP30/GNL KO mice maintained in an AA-insufficient condition. Livers were collected from male SMP30/GNL KO mice at the ages of 3, 6 and 12 months, and wild-type (WT) mice at the ages of 3, 6, 12 and 24 months. To assess protein oxidation, we measured the content of protein carbonyl, which is a major protein oxidation marker. AA levels were measured by 2,4-dinitrophenylhydrazine method using high-performance liquid chromatography. Livers of SMP30/GNL KO mice had just ∼5% as much AA as those of WT mice from 3 to 12 months-of-age. Protein carbonyl levels in livers from SMP30/GNL KO mice were a significant 1.8- to 2.3-fold higher than those from age-matched WT mice. To establish that the AA-insufficiency caused this difference, we added AA to some drinking water, and examined the effect on AA and protein carbonyl levels in livers from SMP30/GNL KO and WT mice. Livers from SMP30/GNL KO mice given extra AA had a significantly higher content than those from their deprived counterparts. Furthermore, protein carbonyl levels in livers from AA-supplemented SMP30/GNL KO mice were significantly lower than those from the SMP30/GNL KO mice without AA supplementation. However, added AA did not affect the protein carbonyl levels in WT mice. These results strongly suggest that AA plays an important role in preventing protein oxidation in vivo, thus enhancing overall health. Geriatr Gerontol Int 2013; ●●: ●●-●●.
    Geriatrics & Gerontology International 10/2013; · 1.58 Impact Factor
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    ABSTRACT: Rationale: Vitamin C (VC) is a potent antioxidant and essential for collagen synthesis. Objectives: We investigated whether VC-treatment prevents and cures smoke-induced emphysema in senescence marker protein-30 knockout (SMP30-KO) mice, which cannot synthesize VC. Methods: Two smoke-exposure experiments using SMP30-KO were conducted. In the first one (a preventive study), four-month-old mice were received minimal VC (0.0375 g/L) [VC(L)] or physiologically sufficient VC (1.5 g/L) [VC(S)], then were exposed to cigarette smoke or smoke-free air for 2 months. Pulmonary evaluations followed when the mice were 6-months of age. The second, study began after the establishment of smoke-induced emphysema (a treatment study). These mice no longer underwent smoke exposure but received VC(S) or VC(L) treatment for 2 months. Morphometric analysis and measurements of oxidative stress, collagen synthesis, and vascular endothelial growth factor (VEGF) in the lungs were evaluated. Measurements and Main Results: Chronic smoke exposure caused emphysema [29.6% increases of mean linear intercepts (MLI) and 106.5% increases of destructive index (DI) compared with the air-only group] in 6-month-old SMP30-KO mice and this emphysema closely resembled human COPD. Furthermore, smoke-induced emphysema persisted in the VC(L) group after smoking cessation, whereas VC-treatment provided pulmonary restoration [18.5% decrease of MLI and 41.3% decrease of DI compared with VC(L) group]. VC-treatment diminished oxidative stress, increased collagen synthesis, and improved VEGF levels in the lungs. Conclusions: Our results suggest that VC not only prevents smoke-induced emphysema in SMP30-KO mice but also restores emphysematous lungs. Therefore, VC may provide a new therapeutic strategy for treating COPD in humans.
    American Journal of Respiratory Cell and Molecular Biology 09/2013; · 4.15 Impact Factor
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    ABSTRACT: BACKGROUNDAIMS: The senescence marker protein-30 (SMP30) is a 34 kDa protein originally identified in rat liver that shows decreased levels with age. Several functional studies using SMP30 knockout (Smp30(Y/-) ) mice established that SMP30 functions as an antioxidant and protects against apoptosis. To address the potential role of SMP30 in nonalcoholic fatty liver disease (NAFLD) pathogenesis, we established Smp30(Y/-) mice on a Lepr(db/db) background (Lepr(db/db)Smp30(Y/-) mice). RESEARCH DESIGNPRINCIPAL FINDINGS: Male Lepr(db/db)Smp30(Y/-) mice were fed a standard diet (340 kcal/100 g, fat 5.6%) for 16 weeks whereupon the lipid/lipoprotein profiles, hepatic expression of genes related to lipid metabolism and endoplasmic reticulum stress markers were analyzed by HPLC, quantitative RT-PCR and western blotting, respectively. Changes in the liver at a histological level were also investigated. The amount of SMP30 mRNA and protein in livers was decreased in Lepr(db/db)Smp30(Y/+) mice compared with Lepr(db/+)Smp30(Y/+) mice. Compared with Lepr(db/db)Smp30(Y/+) mice, 24 week old Lepr(db/db)Smp30(Y/-) mice showed: i) increased small dense LDL-cho and decreased HDL-cho levels; ii) fatty liver accompanied by numerous inflammatory cells and increased oxidative stress; iii) decreased mRNA expression of genes involved in fatty acid oxidation (PPARα) and lipoprotein uptake (LDLR and VLDLR) but increased CD36 levels; and iv) increased endoplasmic reticulum stress. Our data strongly suggest that SMP30 is closely associated with NAFLD pathogenesis, and might be a possible therapeutic target for NAFLD.
    PLoS ONE 06/2013; 8(6):e65698. · 3.53 Impact Factor
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    ABSTRACT: BACKGROUND: Senescence-accelerated mice (SAM) are a series of mouse strains originally derived from unexpected crosses between AKR/J and unknown mice, from which phenotypically distinct senescence-prone (SAMP) and -resistant (SAMR) inbred strains were subsequently established. Although SAMP strains have been widely used for aging research focusing on their short life spans and various age-related phenotypes, such as immune dysfunction, osteoporosis, and brain atrophy, the responsible gene mutations have not yet been fully elucidated.
    BMC Genomics 04/2013; 14(1):248. · 4.04 Impact Factor
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    ABSTRACT: PURPOSE: The effect of an AA deficiency on catecholamine biosynthesis in adult mice in vivo is unknown. Therefore, we quantified catecholamine and the expression of catecholamine synthetic enzymes in the adrenal glands of senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice placed in an AA-deficient state. METHODS: At 30 days of age, mice were divided into the following 4 groups: AA (-) SMP30/GNL KO, AA (+) SMP30/GNL KO, AA (-) wild type (WT), and AA (+) WT. The AA (+) groups were given water containing 1.5 g/L AA, whereas the AA (-) groups received water without AA until the experiment ended. In addition, all mice were fed an AA-depleted diet. Catecholamine levels were measured by a liquid chromatographic method. Tyrosine hydroxylase, dopa decarboxylase, dopamine β-hydroxylase, and phenylethanolamine N-methyltransferase mRNA expression levels were measured with the quantitative real-time polymerase chain reaction (qPCR). Tyrosine hydroxylase and dopamine β-hydroxylase protein levels were quantified by Western blot analysis. RESULTS: In the adrenals of AA (-) SMP30/GNL KO mice, noradrenaline and adrenaline levels decreased significantly compared to other three groups of mice, although there were no significant differences in dopamine β-hydroxylase or phenylethanolamine N-methyltransferase mRNA content. Moreover, there was no significant difference in their dopamine β-hydroxylase protein levels. On the other hand, AA depletion did not affect dopamine levels in adrenal glands of mice. CONCLUSION: An AA deficiency decreases the noradrenaline and adrenaline levels in adrenal glands of mice in vivo.
    European Journal of Nutrition 03/2013; · 3.84 Impact Factor
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    ABSTRACT: Background:Despite the acknowledged importance of ascorbic acid (AA) in maintaining pregnancy and normal fetal development, this vitamin's precise actions remain obscure. Therefore, we investigated the impact of maternal AA content on the growth of fetal mice during the gestation period by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize AA in vivo.Methods:SMP30/GNL KO mice gave birth after a gestation period with absent, low, or normal AA intake. AA was measured by using high performance liquid chromatography and electrochemical detection. Whole body sections were stained with hematoxylin and eosin, Elastica van Gieson, and Azan.Results:The mothers absent AA intake failed to bear young at all because of incomplete fetal development. Offspring born under the low AA condition generally died within a few days after birth. Morphological analysis revealed that the latter neonates of SMP30/GLN KO mothers whose intake of AA was low during gestation manifested abnormal cardiac dilation, congestion of liver and lung, incompletely expanded pulmonary alveoli, and impaired vertebral bodies. In contrast, a normal AA diet produced healthy progeny.Conclusion:A diet sufficiently replete with AA is essential during the gestational period for normal tissue development in the fetus and neonate.Pediatric Research (2013); doi:10.1038/pr.2013.22.
    Pediatric Research 02/2013; · 2.84 Impact Factor
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    ABSTRACT: The senescence marker protein-30 (SMP30), which is also called regucalcin, exhibits gluconolactonase (GNL) activity. Biochemical and biological analyses revealed that SMP30/GNL catalyzes formation of the γ-lactone-ring of l-gulonate in the ascorbic acid biosynthesis pathway. The molecular basis of the γ-lactone formation, however, remains elusive due to the lack of structural information on SMP30/GNL in complex with its substrate. Here, we report the crystal structures of mouse SMP30/GNL and its complex with xylitol, a substrate analogue, and those with 1,5-anhydro-d-glucitol and d-glucose, product analogues. Comparison of the crystal structure of mouse SMP30/GNL with other related enzymes has revealed unique characteristics of mouse SMP30/GNL. First, the substrate-binding pocket of mouse SMP30/GNL is designed to specifically recognize monosaccharide molecules. The divalent metal ion in the active site and polar residues lining the substrate-binding cavity interact with hydroxyl groups of substrate/product analogues. Second, in mouse SMP30/GNL, a lid loop covering the substrate-binding cavity seems to hamper the binding of l-gulonate in an extended (or all-trans) conformation; l-gulonate seems to bind to the active site in a folded conformation. In contrast, the substrate-binding cavities of the other related enzymes are open to the solvent and do not have a cover. This structural feature of mouse SMP30/GNL seems to facilitate the γ-lactone-ring formation.
    PLoS ONE 01/2013; 8(1):e53706. · 3.53 Impact Factor
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    ABSTRACT: Senescence marker protein-30 (SMP30) was first described as a physiologic entity that decreases in the rat liver and kidney with aging. Previously, we established that SMP30 is the lactone-hydrolyzing enzyme gluconolactonase (GNL), which is involved in ascorbic acid (AA) biosynthesis. In the present study, we found SMP30/GNL mRNA expressed in the mouse ovary. To ascertain the reason for ovarian SMP30/GNL expression, we examined mice during gestation. SMP30/GNL mRNA expression was evident at the start of gestation, increased for the next eight days then decreased rapidly. Moreover, L-gulono-γ-lactone oxidase (Gulo) mRNA, which catalyzes the last step of AA, was found in the ovaries of these mice. The variations of these genes' expression showed an inverse pattern to that of Cyp19a1 (aromatase) mRNA expression. Therefore, the SMP30/GNL and Gulo mRNA expression might be regulated by estrogen levels in the ovary. Since the presence of both SMP30/GNL and Gulo mRNAs could indicate that AA synthesis occurs in the ovary, we quantified AA levels in mouse ovaries during gestation. However, no correlation was found between changes of AA content and SMP30/GNL or Gulo mRNAs expression at this site. Moreover, we compared the changes of AA content during gestation between wild-type and SMP30/GNL knockout mice, which cannot synthesize AA, and found no significant differences between them. These results indicated that, although AA synthesis might occur in the ovaries, the amount of AA which is synthesized in ovaries must be quite low and insufficient to influence the AA content in ovary.
    Biological & Pharmaceutical Bulletin 01/2013; 36(12):2005-8. · 1.85 Impact Factor
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    ABSTRACT: Ascorbic acid (AA) functions as an electron donor and scavenges reactive oxygen species such as superoxide, singlet oxygen, and hydroxyl radicals in vitro. However, little is known about the effect of an AA deficiency on protein and lipid oxidation levels in the liver. Therefore, we measured the levels of protein carbonyl and thiobarbituric acid reactive substances (TBARS) in livers from senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are deficient in AA, because they lack the SMP30/GNL gene, which is essential for the biosynthesis of AA in vivo. To track the effect of an AA deficiency, at 30 d of age, mice were divided into the following four groups: AA (-) SMP30/GNL KO, AA (+) SMP30/GNL KO, AA (-) wild type (WT), and AA (+) WT. The AA (+) groups were given water containing 1.5 g/L AA, whereas the AA (-) groups received water without AA for 57 d. All mice were fed an AA-free diet. Subsequently, protein carbonyl levels in livers from AA (-) SMP30/GNL KO mice were significantly higher than those from the other three groups; however, TBARS levels were not significantly different among the four groups. Therefore, AA must act as an anti-oxidant for proteins but might not directly protect lipid oxidation in the liver.
    Journal of Nutritional Science and Vitaminology 01/2013; 59(6):489-95. · 0.99 Impact Factor
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    ABSTRACT: Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24 hours for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1 / type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.
    Biochemical and Biophysical Research Communications 12/2012; · 2.28 Impact Factor
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    ABSTRACT: The post-translational citrullination (deimination) process is mediated by peptidylarginine deiminases (PADs), which convert peptidylarginine into peptidylcitrulline in the presence of high calcium concentrations. Over the past decade, PADs and protein citrullination have been commonly implicated as abnormal pathological features in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease and rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs and citrullination in the pathogenesis of prion diseases. Prion diseases (also known as transmissible spongiform encephalopathies) are fatal neurodegenerative diseases that are pathologically well characterized as the accumulation of disease-associated misfolded prion proteins, spongiform changes, glial cell activation and neuronal loss. We previously demonstrated that the upregulation of PAD2, mainly found in reactive astrocytes of infected brains, leads to excessive citrullination, which is correlated with disease progression. Further, we demonstrated that various cytoskeletal and energy metabolism-associated proteins are particularly vulnerable to citrullination. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity and enhanced plasminogen-binding affinity. These findings suggest that PAD2 and citrullinated proteins may play a key role in the brain pathology of prion diseases. By extension, we believe that abnormal increases in protein citrullination may be strong evidence of neurodegeneration.
    Prion 09/2012; 6(5). · 1.97 Impact Factor
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    ABSTRACT: OBJECTIVE: The peptidylarginine deiminase 4 (PAD4) gene and PAD4 autoantibodies have been associated with rheumatoid arthritis (RA) and its pathogenesis. Therefore, methods for accurately determining their levels in the peripheral blood of these patients would be a diagnostic asset. The objective of our study was to adapt the enzyme-linked immunosorbent assay (ELISA) method for evaluating PAD4 levels in human blood. METHODS: We prepared recombinant human (h)PAD1, -2, -3, and -4 proteins to develop mouse monoclonal antibodies specific to hPAD4. We then generated six monoclonal antibodies against hPAD4 and developed two new sandwich ELISA methods for evaluating hPAD4 and PAD4 autoantibodies in the peripheral blood from 32 patients with RA, ten patients with osteoarthrosis, and 20 healthy individuals. RESULTS: The distribution of hPAD4 in the patients' plasma was determined. Two populations were identified: one group with high hPAD4 levels (>0.57 ng/mL) and a second group with near-zero levels (<0.1 ng/mL). Most patients approximating zero hPAD4 levels had PAD4 autoantibodies. In contrast, most of those with higher plasma hPAD4 levels did not have detectable PAD4 autoantibodies. CONCLUSION: The combination of these sandwich ELISA methods may be a potentially beneficial clinical tool for diagnosing RA.
    Modern Rheumatology 09/2012; 23(4). · 2.21 Impact Factor

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3k Citations
580.15 Total Impact Points


  • 1987–2014
    • Tokyo Metropolitan Institute of Gerontology
      Edo, Tōkyō, Japan
  • 2008–2012
    • Hallym University
      • College of Medicine
      Seoul, Seoul, South Korea
  • 2011
    • Tokyo Metropolitan University
      • Graduate School of Science and Engineering
      Edo, Tōkyō, Japan
  • 2008–2010
    • Toho University
      • Department of Biochemistry
      Edo, Tōkyō, Japan
  • 2009
    • Kyoto Prefectural University of Medicine
      • Division of Endocrinology, Diabetes and Metabolism
      Kioto, Kyōto, Japan
  • 2004–2007
    • Kobe Pharmaceutical University
  • 2006
    • Niigata University
      • Division of Neurophysiology
      Niahi-niigata, Niigata, Japan
  • 2004–2006
    • Pusan National University
      • College of Pharmacy
      Pusan, Busan, South Korea
  • 1997–2006
    • Juntendo University
      • Department of Medicine
      Tokyo, Tokyo-to, Japan
    • Chiba University
      Tiba, Chiba, Japan
  • 2002
    • The University of Tokyo
      • Faculty & Graduate School of Medicine
      Tokyo, Tokyo-to, Japan
  • 1991–1998
    • The Jikei University School of Medicine
      • Department of Internal Medicine
      Tokyo, Tokyo-to, Japan
  • 1994–1996
    • Saitama Medical University
      • Department of Internal Medicine
      Saitama, Saitama-ken, Japan