Detection of a glutathionyl-carbonylated group (GS-CO-) on D-dopachrome tautomerase with preferential binding of GS-CO- to MIF proteins in rat livers damaged by carbon tetrachloride.

Liver damage has been induced in animal experiments using carbon tetrachloride (CCl4), a potent hepatotoxin. CCl4 is activated by cytochrome P450 2E1, which results in the formation of various metabolites including phosgene. Although D-dopachrome tautomerase (DDT) is abundant in the liver, its role currently remains unclear. The biological activity of DDT, for which the N-terminal proline is a key site, has been detected in various tissues. We herein incidentally detected a 333 Da modification to the N-terminal proline of DDT in rat livers damaged by CCl4. We identified that this modification as glutathionyl carbonylated group, which was formed by condensation of phosgene and reduced glutathione (GSH). We examined other glutathionyl-carbonylated proteins using two dimensional-polyacrylamide gel electrophoresis, mass spectrometry, and Western blotting for GSH, and detected only one glutathionyl-carbonylated protein, macrophage migration inhibitory factor (MIF). DDT belongs to the MIF family of proteins, and amino acid sequence identity between DDT and MIF is 33%. We concluded that MIF family proteins are major targets for glutathionyl carbonylation.

Decrease in Glycosaminoglycan with Aging in Normal Rat Articular Cartilage Is Greater in Females than in Males.

OBJECTIVE: Osteoarthritis (OA) is more prevalent in females. We hypothesized that changes in articular cartilage (AC) constituents with aging may cause differences. Herein, we aimed to compare the changes in AC constituents with aging in male and female normal rats. DESIGN: The glycosaminoglycan (GAG) and collagen (COL) contents of the AC in knee, hip, and shoulder joints of male and female rats were quantified and compared between age groups and sexes. RESULTS: The amount of GAG was decreased in multiple joints in both males and females with aging. In females, it had a significant decrease in all joints measured. The decrease in GAG with aging was more severe in females than in males. Even in young rats, the amount of knee joint GAG was significantly less in females than in males. The amount of COL in the AC was unchanged with aging in both sexes. CONCLUSIONS: The drastic GAG decrease with aging in female normal rats may explain the higher prevalence and more severe OA in females.

Effect of chronic administration with human thioredoxin-1 transplastomic lettuce on diabetic mice.

SCOPE: Human thioredoxin-1 (hTrx-1) is a defensive protein induced by various stresses and exerts antioxidative and anti-inflammatory effects. Previously, we described a transplastomic lettuce overexpressing hTrx-1 that exerts a protective effect against oxidative damage in a pancreatic ß-cell line. In this study, we treated diabetic mice (Akita mice) with exogenous hTrx-1 and evaluated the effects. METHODS AND RESULTS: Treatment with drinking water and single applications of exogenous hTrx-1 did not influence the feeding, drinking behavior, body weight, blood glucose, or glycosylated hemoglobin (HbA1c) levels in Akita mice. However, chronic administration of a 10% hTrx-1 lettuce-containing diet was associated with a significant reduction from the baseline of HbA1c levels compared with mice fed a wild-type lettuce-containing diet. It also resulted in an increased number of goblet cells in the small intestine, indicating that mucus was synthesized and secreted. CONCLUSION: Our results revealed that the administration of an hTrx-1 lettuce-containing diet improves the baseline level of HbA1c in Akita mice. This effect is mediated through goblet cell proliferation and possibly related to protection against postprandial hyperglycemia by mucus, which results in the improvement of blood glucose control. These findings suggest that the hTrx-1 lettuce may be a useful tool for the continuous antioxidative and antidiabetic efficacies of the hTrx-1 protein.

Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry.

NAD+ is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD+-degrading enzymes in mammals. Numerous studies report that tissue NAD+ levels decrease during aging and age-related diseases and suggest that NAD+ replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD+ levels only 40-60% in mammalian cells. Mechanisms underlying the limited increases in NAD+ levels remain to be determined. Here we show that Nampt is inhibited in cells and that enhanced expression of Nampt activates NAD+ breakdown. Combined with the measurement of each cell's volume, we determined absolute values (µM/h) of the rates of NAD+ synthesis (RS) and breakdown (RB) using a flux assay with a 2H (D)-labeled Nam, together with the absolute NAD+ concentrations in various mammalian cells including primary cultured cardiomyocytes under the physiological conditions and investigated the relations among total cellular Nampt activity, RS, RB, and the NAD+ concentration. NAD+ concentration was maintained within a narrow range (400-700 µM) in the cells. RS was much smaller than the total Nampt activity, indicating that NAD+ synthesis from Nam in the cells is suppressed. Forced expression of Nampt leading to 6-fold increase in total Nampt activity induced only a 1.6-fold increase in cellular NAD+ concentration. Under the conditions, RS increased by 2-fold, while 2-fold increase in RB was also observed. The small increase in cellular NAD+ concentration is likely due to both inhibited increase in the NAD+ synthesis and the activation of its breakdown. Our findings suggest that cellular NAD+ concentrations do not vary dramatically by the physiological fluctuation of Nampt expression and show the tight link between the NAD+ synthesis and its breakdown.

Complete solubilization of cartilage using the heat-stable protease thermolysin for comprehensive GAG analysis.

Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70 °C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50 °C. Overnight digestion with thermolysin completely solubilized cartilage, whereas collagenase with >10-times higher proteolytic activity digested <20% of collagen. Following thermolysin treatments, almost all GAGs were extracted from the cartilage, whereas only 56% of GAGs were extracted after collagenase digestion. Disaccharide analyses of extracted GAG chains revealed >98% extraction efficiencies of several GAG classes from thermolysin-treated cartilage, compared with <60% extraction efficiencies using collagenase, depending on GAG classes. These results indicate that thermolysin allows complete GAG extraction from solid articular cartilage and that complete solubilization is required for accurate and reproducible analyses of cartilage GAGs. Hence, thermolysin offers a tool for complete solubilization of cartilage prior to comprehensive GAGomic analysis, and is likely applicable to other collagen-rich tissues such as ligaments, skin, and blood vessels.

Association of plasma free amino acids with hyperuricemia in relation to diabetes mellitus, dyslipidemia, hypertension and metabolic syndrome.

Previous studies demonstrated independent contributions of plasma free amino acids (PFAAs) and high uric acid (UA) concentrations to increased risks of lifestyle-related diseases (LSRDs), but the important associations between these factors and LSRDs remain unknown. We quantified PFAAs and UA amongst Japanese subjects without LSRDs (no-LSRD, n = 2805), and with diabetes mellitus (DM, n = 415), dyslipidemia (n = 3207), hypertension (n = 2736) and metabolic syndrome (MetS, n = 717). The concentrations of most amino acids differed significantly between the subjects with and without hyperuricemia (HU) and also between the no-LSRD and LSRD groups (p < 0.05 to 0.001). After adjustment, the logistic regression analyses revealed that lysine in DM, alanine, proline and tyrosine in dyslipidemia, histidine, lysine and ornithine in hypertension, and lysine and tyrosine in MetS demonstrated significant positive associations with HU among the patients with LSRDs only (p < 0.05 to 0.005). By contrast, arginine, asparagine and threonine showed significant inverse associations with HU in the no-LSRD group only (p < 0.05 to 0.01). For the first time, we provide evidence for distinct patterns of association between PFAAs and HU in LSRDs, and postulate the possibility of interplay between PFAAs and UA in their pathophysiology.

Alteration in plasma free amino acid levels and its association with gout.

BACKGROUND: Studies on the association of plasma-free amino acids with gout are very limited and produced conflicting results. Therefore, we sought to explore and characterize the plasma-free amino acid (PFAA) profile in patients with gout and evaluate its association with the latter. METHODS: Data from a total of 819 subjects (including 34 patients with gout) undergoing an annual health examination program in Shimane, Japan were considered for this study. Venous blood samples were collected from the subjects and concentrations of 19 plasma amino acids were determined by high-performance liquid chromatography-electrospray ionization-mass spectrometry. Student's t-test was applied for comparison of variables between patient and control groups. The relationships between the presence or absence of gout and individual amino acids were investigated by logistic regression analysis controlling for the effects of potential demographic confounders. RESULTS: Among 19 amino acids, the levels of 10 amino acids (alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, tryptophan, valine) differed significantly (P < .001 to .05) between the patient and control groups. Univariate logistic regression analysis revealed that plasma levels of alanine, isoleucine, leucine, phenylalanine, tryptophan and valine had significant positive associations (P < .005 to .05) whereas glycine and serine had significant inverse association (P < .05) with gout. CONCLUSIONS: The observed significant changes in PFAA profiles may have important implications for improving our understanding of pathophysiology, diagnosis and prevention of gout. The findings of this study need further confirmation in future large-scale studies involving a larger number of patients with gout.

Plasma free amino acid profiles evaluate risk of metabolic syndrome, diabetes, dyslipidemia, and hypertension in a large Asian population.

BACKGROUND: Recently, the association of plasma free amino acid (PFAA) profile and lifestyle-related diseases has been reported. However, few studies have been reported in large Asian populations, about the usefulness of PFAAs for evaluating disease risks. We examined the ability of PFAA profiles to evaluate lifestyle-related diseases in so far the largest Asian population. METHODS: We examined plasma concentrations of 19 amino acids in 8589 Japanese subjects, and determined the association with variables associated with obesity, blood glucose, lipid, and blood pressure. We also evaluated the PFAA indexes that reflect visceral fat obesity and insulin resistance. The contribution of single PFAA level and relevant PFAA indexes was also examined in the risk assessment of lifestyle-related diseases. RESULTS: Of the 19 amino acids, branched-chain amino acids and aromatic amino acids showed association with obesity and lipid variables. The PFAA index related to visceral fat obesity showed relatively higher correlation with variables than that of any PFAA. In the evaluation of lifestyle-related disease risks, the odds ratios of the PFAA index related to visceral fat obesity or insulin resistance with the diseases were higher than most of those of individual amino acid levels even after adjusting for potential confounding factors. The association pattern of the indexes and PFAA with each lifestyle-related disease was distinct. CONCLUSIONS: We confirmed the usefulness of PFAA profiles and indexes as markers for evaluating the risks of lifestyle-related diseases, including diabetes mellitus, metabolic syndrome, dyslipidemia, and hypertension in a large Asian population.

Human thioredoxin-1 attenuates the rate of lipopolysaccharide-induced preterm delivery in mice in association with its anti-inflammatory effect.

BACKGROUND: Maternal intrauterine infection/inflammation represents the major etiology of preterm delivery and the leading cause of neonatal mortality and morbidity. The aim of this study was to investigate the anti-inflammatory properties of thioredoxin-1 in vivo and its potential ability to attenuate the rate of inflammation-induced preterm delivery. METHODS: Two intraperitoneal injections of lipopolysaccharide from Escherichia coli were administered in pregnant mice on gestational day 15, with a 3-h interval between the injections. From either 1 h before or 1 h after the first lipopolysaccharide injection, mice received three intravenous injections of either recombinant human thioredoxin-1, ovalbumin, or vehicle, with a 3-h interval between injections. RESULTS: Intraperitoneal injection of lipopolysaccharide induced a rise of tumor necrosis factor-α, interferon-γ, monocyte chemotactic protein 1, and interleukin-6 in maternal serum levels and provoked preterm delivery. Recombinant human thoredoxin-1 prevented the rise in these proinflammatory cytokine levels. After the inflammatory challenge, placentas exhibited severe maternal vascular dilatation and congestion and a marked decidual neutrophil activation. These placental pathological findings were ameliorated by recombinant human thioredoxin-1, and the rate of inflammation-induced preterm delivery was attenuated. CONCLUSION: Thioredoxin-1 may thus represent a novel effective treatment to delay inflammation-induced preterm delivery.

Association between feeling upon awakening and use of information technology devices in Japanese children.

BACKGROUND: The objective of this study was to clarify the relationship between feeling upon awakening (FA) and time spent using information technology (IT) devices by children in kindergartens, elementary schools, and junior high schools in Shimane, Japan. METHODS: In October 2008, a self-report survey was distributed to 2075 children in kindergartens (n = 261), elementary schools (n = 1162), and junior high schools (n = 652) in Shimane, Japan. The questionnaire gathered data on sex, school year, feeling upon awakening, and time spent using IT devices after school (television, videos on television, video games, personal computers, and cellular phones). After adjusting for sex and school year, data were analyzed by multivariate logistic regression analysis to calculate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: A total of 2030 children completed this survey (response rate, 97.8%). Negative FA was associated with watching television more than 2 hours/day (OR = 1.51, 95% CI = 1.23-1.85), playing video games more than 30 minutes/day (1.50, 1.20-1.87), and using personal computers more than 30 minutes/day (1.35, 1.04-1.75). CONCLUSIONS: Time spent using IT devices affected the FA of children in kindergarten through junior high school. We propose the development of guidelines regarding the appropriate amount of time this population should spend using IT devices.

Association analysis of the NOD2 gene with susceptibility to graft-versus-host disease in a Japanese population.

Members of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family participate in the innate immune system, exerting widespread effects on cytokine secretion, autophagy, and apoptosis. Recent studies in Caucasians revealed the association between mutants of NOD2, a member of the NLR family, and severity of acute graft-versus-host disease (GVHD). NOD2 polymorphism screening has been recommended for donor selection and risk assessment at bone marrow transplantation. To investigate whether NOD2 plays a role in the pathogenesis of GVHD in a Japanese population, we examined DNA from 142 bone marrow transplant patient/donor pairs to detect genetic variation in the NOD2 gene. No genetic variants of NOD2 were associated with the severity of acute GVHD in our patients. However, a weak association between a single nucleotide polymorphism in the NOD2 gene (R471C) and acute myeloid leukemia in the bone marrow patients (p = 0.029, odds ratio 4.08, 95% CI 1.22-13.67) was detected. This polymorphism was not prevalent in 479 Crohn's disease (CD) patients in Japan. These results suggest that, in the Japanese population, unlike the Caucasian, NOD2 is not a major contributor to susceptibility to severe acute GVHD.

Mechanism of skull suture maintenance and interdigitation.

Skull sutures serve as growth centers whose function involves multiple molecular pathways. During periods of brain growth the sutures remain thin and straight, later developing complex fractal interdigitations that provide interlocking strength. The nature of the relationship between the molecular interactions and suture pattern formation is not understood. Here we show that by classifying the molecules involved into two groups, stabilizing factors and substrate molecules, complex molecular networks can be modeled by a simple two-species reaction-diffusion model that recapitulates all the known behavior of suture pattern formation. This model reproduces the maintenance of thin sutural tissue at early stages, the later modification of the straight suture to form osseous interdigitations, and the formation of fractal structures. Predictions from the model are in good agreement with experimental observations, indicating that the model captures the essential nature of the interdigitation process.

Thioredoxin, an anti-oxidant protein, protects mouse embryos from oxidative stress-induced developmental anomalies.

During the early postimplantation period, rodent embryos survive in a relatively anaerobic environment in utero and are vulnerable to a high oxygen pressure. They become resistant to oxygen stress when they are exposed to a higher oxygen pressure after the uteroplacental circulation is established. However, it is unknown how embryos acquire such resistance against oxidative stress. This study was undertaken to examine whether an antioxidant protein thioredoxin (TRX) plays a significant role in the embryonic acquisition of the tolerance to oxidative stress. E7.5 embryos of C57BL/6 wild-type (WT) and human TRX (hTRX) inserted-transgenic (Tg) embryos were cultured under 10 or 25% O2 and their growth and morphological differentiation were evaluated. The TRX expression and the products of oxidative stress (8-hydroxy-2'-deoxy-guanosine and carbonylated proteins) in their tissues were also examined. When WT embryos were cultivated in vitro under 25% O2, their growth was significantly disturbed and various developmental abnormalities were induced, which did not occur in embryos grown under 10% O2. However, such embryotoxic effects of oxygen were significantly attenuated in the hTRX Tg embryos that continuously express hTRX. Accumulation of the products of oxidative stress was significantly reduced in hTRX Tg embryos as compared with that in WT embryos. The TRX transgene appears to provide the embryo with the resistance against oxidative stress and may play a crucial role in the redox regulation in embryos.