Naoki Maruyama

Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts.

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 24h 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.

Vitamin C Prevents Cigarette Smoke–Induced Pulmonary Emphysema in Mice and Provides Pulmonary Restoration

Vitamin C (VC) is a potent antioxidant and is essential for collagen synthesis. We investigated whether VC treatment prevents and cures smoke-induced emphysema in senescence marker protein-30 knockout (SMP30-KO) mice, which cannot synthesize VC. Two smoke-exposure experiments using SMP30-KO mice were conducted. In the first one (a preventive study), 4-month-old mice received minimal VC (0.0375 g/l) [VC(L)] or physiologically sufficient VC (1.5 g/l) [VC(S)] and 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 was performed, and measurements of oxidative stress, collagen synthesis, and vascular endothelial growth factor in the lungs were evaluated. Chronic smoke exposure caused emphysema (29.6% increases of mean linear intercepts [MLI] and 106.5% increases of destructive index compared with the air-only group) in 6-month-old SMP30-KO mice, and this emphysema closely resembled human chronic obstructive pulmonary disease. 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 destructive index compared with VC(L) group). VC treatment diminished oxidative stress, increased collagen synthesis, and improved vascular endothelial growth factor levels in the lungs. 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 chronic obstructive pulmonary disease in humans.

Immune Abnormalities Induced by Human Endogenous Retroviral Peptides: With Reference to the Pathogenesis of Systemic Lupus Erythematosus

P15E is a specific sequence among the envelope gene (env)-encoded transmembrane proteins of exogenous and endogenous retroviruses. A synthetic peptide (CKS-17) that shows homology to this p15E region in several species of retrovirus is known to induce immune abnormalities. In this study, we examined the effect of a synthetic peptide derived from a region of human endogenous retrovirus (HERV) clone 4-1 (λ 4-1) similar to sequences of CKS-17 on the induction of systemic lupus erythematosus (SLE)-related immune abnormalities. Our results indicated that this peptide could induce T-cell activation and anergy in normal peripheral blood mononuclear cells, and the peptide could also promote the production of interleukins IL-6 and IL-16. These phenomena are representative immune abnormalities observed in SLE patients. Thus, our findings support the possibility that HERV acts as a pathogen in human SLE.

Effect of vitamin C depletion on UVR-B induced cataract in SMP30/GNL knockout mice.

We investigated whether decreased vitamin C (VC) in a mouse model increases lens opacity (cataract) induced by inxa0vivo exposure to ultraviolet radiation type B (UVR-B). Senescence marker protein-30 (SMP30) knockout (KO) mice, which cannot synthesize VC due to genetic disruption of the gluconolactonase (GNL) gene, were divided into 2 groups: VC sufficient (VC (+)) and VC deficient (VC (-)). Starting at 1 month of age, these groups had free access to water containing 0.0375 and 1.5xa0g/L of VC, respectively. SMP30 KO VC (-), SMP30 KO VC (+), and wild-type (WT) mice, all 14 weeks of age, were unilaterally exposed inxa0vivo to UVR-B (200xa0mW/cm(2)) for 100xa0s twice a week for 3 weeks (total: 1200xa0mJ/cm(2)). At 48xa0h after the last UVR-B exposure, cataract morphology was documented, and the ratio of cataract induction was quantified as the cataract area ratio (opacity area/anterior capsule). UVR-B exposure induced cataract mainly at anterior sub-capsular in SMP30 KO VC (-), SMP30 KO VC (+), and WT mice. In SMP30 KO VC (-) lenses the opacities were more extensive than in SMP30 KO VC (+) or WT lenses (cataract area ratios: 59.3%xa0±xa010% vs. 32.2%xa0±xa011.7% or 29.0%xa0±xa09.0%; Pxa0<xa00.01). In conclusion, VC depletion may increase the susceptibility to develop UVR-B induced cataracts in mice unable to endogenously produce VC.

Sequence Analysis of Human Endogenous Retrovirus Clone 4-1 in Systemic Lupus Erythematosus

Human endogenous retroviruses (HERV) have emerged as a possible cause of systemic lupus erythematosus (SLE). We previously detected serum antibodies to the gag region of HERV clone 4-1 in patients with SLE, but not in normal volunteers. In the present study, we detected clone 4-1 messenger RNA (mRNA) in peripheral blood mononuclear cells (PBMC) from SLE patients and performed sequence analysis of the cDNA or genomic DNA from clone 4-1 in these patients. Clone 4-1 mRNA was detected in all of the SLE patients tested, although it was not found in normal controls. Sequence analysis of clone 4-1 in these SLE patients revealed inactivation of the stop codons in part of the gag region. In addition, a computer search of current sequence libraries revealed that the clone 4-1 gag genomic DNA from SLE patients was more highly homologous with the clone 4-1 sequence in chromosome 11 from normal individuals when compared with the sequence of clone 4-1 integrated in the other chromosomes. It is possible that transcription of clone 4-1 from chromosome 11 occurs in SLE, and that the stop codon inactivation contributes to the translation of clone 4-1 gag proteins in patients with this disease

Bioavailability of vitamin C from mashed potatoes and potato chips after oral administration in healthy Japanese men.

Potato (Solanum tuberosum) tubers contain vitamin C (VC) and commercial potato chips have more VC content per wet weight by dehydration during frying. However, intestinal absorption of VC from orally ingested potatoes and its transfer to the blood remains questionable. The present study was designed to determine whether the dietary consumption of potatoes affects VC concentration in plasma and urinary excretion of VC in human subjects. After overnight fasting, five healthy Japanese men between 22 and 27 years of age consumed 87 g mashed potatoes and 282 g potato chips. Each portion contained 50 mg of VC, 50 mg VC in mineral water and mineral water. Before and after a single episode of ingestion, blood and urine samples were collected every 30 min or 1 h for 8 h. When measured by subtraction of the initial baseline value before administration of potatoes from the values measured throughout the 8 h test period, plasma VC concentrations increased almost linearly up to 3 h. Subsequently, the values of potato-fed subjects were higher than those of water, but did not differ significantly from those of VC in water (P = 0·14 and P = 0·5). Less VC tended to be excreted in urine during the 8 h test than VC in water alone (17·0 (sem 7·5) and 25·9 (sem 8·8) v. 47·9 (sem 17·9) μmol/mmol creatinine). Upon human consumption, mashed potatoes and potato chips provide VC content that is effectively absorbed in the intestine and transferred to the blood. Clearly, potatoes are a readily available source of dietary VC.

A Significant Relationship between Plasma Vitamin C Concentration and Physical Performance among Japanese Elderly Women

BACKGROUNDnMaintenance of physical performance could improve the quality of life in old age. Recent studies suggested a beneficial relationship between antioxidant vitamin (eg, vitamin C) intake and physical performance in elderly people. The purpose of this study was to examine the relationship between plasma vitamin C concentration and physical performance among Japanese community-dwelling elderly women.nnnMETHODSnThis is a cross-sectional study involving elderly females residing in an urban area in Tokyo, Japan, in October 2006. We examined anthropometric measurements, physical performance, lifestyles, and plasma vitamin C concentration of participants.nnnRESULTSnA total of 655 subjects who did not take supplements were analyzed. The mean age (±standard deviation) of participants was 75.7 ± 4.1 years in this study. The geometric mean (geometric standard deviation) of plasma vitamin C concentration was 8.9 (1.5) μg/mL. The plasma vitamin C concentration was positively correlated with handgrip strength, length of time standing on one leg with eyes open and walking speed, and inversely correlated with body mass index. After adjusting for the confounding factors, the quartile plasma vitamin C level was significantly correlated with the subjects handgrip strength (p for trend = .0004) and ability to stand on one leg with eyes open (p for trend = .049).nnnCONCLUSIONSnIn community-dwelling elderly women, the concentration of plasma vitamin C related well to their muscle strength and physical performance.

Potato chip intake increases ascorbic acid levels and decreases reactive oxygen species in SMP30/GNL knockout mouse tissues.

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, this study 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 the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, eyeball, and epididymal fat compared with AA-depleted mice. The 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.

Pancreatic insulin release in vitamin C-deficient senescence marker protein-30/gluconolactonase knockout mice

We recently identified senescence marker protein-30 as the lactone-hydrolyzing enzyme gluconolactonase, which is involved in vitamin C biosynthesis. In this study, we investigated the effects of vitamin C on insulin secretion from pancreatic β-cells using senescence marker protein-30/gluconolactonase knockout mice. In intraperitoneal glucose tolerance tests, vitamin C-deficient senescence marker protein-30/gluconolactonase knockout mice demonstrated impaired glucose tolerance with significantly lower blood insulin levels at 30 and 120 min post-challenge than in wild type mice (p<0.01–0.05). In contrast, vitamin C-sufficient senescence marker protein-30/gluconolactonase knockout mice demonstrated significantly higher blood glucose and lower insulin only at the 30 min post-challenge time point (p<0.05). Senescence marker protein-30/gluconolactonase knockout mice showed enhanced insulin sensitivity regardless of vitamin C status. Static incubation of islets revealed that 20 mM glucose-stimulated insulin secretion and islet ATP production were significantly decreased at 60 min only in vitamin C-deficient SMP30/GNL knockout mice relative to wild type mice (p<0.05). These results indicate that the site of vitamin C action lies between glycolysis and mitochondrial oxidative phosphorylation, while SMP30 deficiency itself impairs the distal portion of insulin secretion pathway.

Age-related changes of dopamine, noradrenaline and adrenaline in adrenal glands of mice

Aim:u2003 Catecholamines, which are physiologically important neurotransmitters and hormones, apparently decrease in the brain and plasma as some species age. Because this observation has engendered controversy, we used mice to investigate whether age‐related changes occur in adrenal catecholamine levels and in the expression of catecholamine synthetic enzymes.