Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine.

Regenerative medicine has received a lot of attention as a novel strategy for injuries and diseases that are difficult to cure using current techniques. Cell production, which is vital for regenerative medicine, has undergone remarkable progress via breakthroughs in developmental biology and tissue engineering; currently, cell production requires numerous experimental operators performing manual, small-scale cell cultures. Other major obstacles for cell production and regenerative medicine include the variable quality of products based on the experimental procedure, the skills of operators, the level of labor required for production, and costs. Technological developments are required to overcome this, including automation instead of manual culture. Age-related macular regeneration (AMD) is a refractory ocular disease that causes severe deterioration in central vision due to senescence in the retinal pigment epithelium (RPE). Recently, we performed an autologous transplantation of induced pluripotent stem (iPS) cell-derived RPE cell sheets and started clinical research on allografts from RPE cell suspensions differentiated from iPS cells. The use of regenerative therapies for AMD using iPS cell-derived RPE is expected to become more widespread. In the present study, human iPS cell-derived RPE cells were cultured to form RPE cell sheets using equipment with a closed culture module. The quality of the automated cultured RPE cell sheets was confirmed by comparing their morphological and biological properties with those of manually generated RPE cell sheets. As a result, machine-cultured RPE sheets displayed the same quality as manually cultured RPE sheets, showing that iPS cell-derived RPE cell sheets were successfully cultured by an automated process.

Sodium-glucose co-transporter type 2 inhibitors reduce evening home blood pressure in type 2 diabetes with nephropathy.

BACKGROUND: The effects of sodium-glucose co-transporter type 2 inhibitors on home blood pressure were examined in type 2 diabetes with nephropathy. METHODS: The patients with diabetic nephropathy were screened from medical records in our hospitals. Among them, 52 patients who measured home blood pressure and started to take sodium-glucose co-transporter type 2 inhibitors were selected. Clinical parameters including estimated glomerular filtration rate, albuminuria and home blood pressure for 6 months were analysed. RESULTS: Sodium-glucose co-transporter type 2 inhibitors (luseogliflozin 5 mg/day or canagliflozin 100 mg/day) reduced body weight, HbA1c, albuminuria, estimated glomerular filtration rate and office blood pressure. Although sodium-glucose co-transporter type 2 inhibitors did not alter morning blood pressure, it reduced evening systolic blood pressure. Regression analyses revealed that decreases in evening blood pressure predicted decrements in albuminuria. CONCLUSION: The present data suggest that sodium-glucose co-transporter type 2 inhibitors suppress sodium overload during daytime to reduce evening blood pressure and albuminuria.

Involvement of visceral fat in the pathogenesis of albuminuria in patients with type 2 diabetes with early stage of nephropathy.

BACKGROUND: Visceral obesity has been implicated in the pathogenesis of diabetic nephropathy. Waist circumference has been used as a surrogate measure of visceral fat mass; however, subcutaneous fat mass is also correlated with waist circumference. We therefore conducted this cross-sectional study to clarify the relationship between directly measured sizes of visceral and subcutaneous fat and microalbuminuria in patients with type 2 diabetes (T2DM). METHODS: We studied a total of 208 adult Japanese individuals with T2DM, 99 women and 109 men, with a mean +/- standard deviation (SD) age of 56 +/- 13 years. Patients with macroalbuminuria, defined as a urinary albumin-to-creatinine ratio (ACR) >or=300 mg/g creatinine, and those with an estimated glomerular filtration rate <15 ml/min/1.73 m(2) were excluded. Visceral and subcutaneous fat areas were measured by abdominal computed tomography. RESULTS: In the univariate correlational analysis, logarithmically transformed urinary ACR was significantly associated with visceral fat area (r = 0.14, p = 0.047) but not with subcutaneous fat area (r = 0.08, p = 0.237). In the multiple regression analysis with stepwise selection procedure, visceral fat area but not subcutaneous fat area was selected as an independent variable that was statistically associated with urinary ACR. CONCLUSION: This cross-sectional study suggests that increased visceral but not subcutaneous fat is independently associated with microalbuminuria in Japanese adult patients with T2DM.

Higher levels of urinary albumin excretion within the normal range predict faster decline in glomerular filtration rate in diabetic patients.

OBJECTIVE To assess the relationship between albuminuria, including elevation within the normal range, and decline in glomerular filtration rate (GFR) in diabetic patients. RESEARCH DESIGN AND METHODS A total of 5,449 Japanese diabetic patients were categorized according to sex and urinary albumin-to-creatinine ratio (ACR; <5, 5-9, 10-29, 30-99, 100-299, 300-999, 1,000-2,999, and > or =3,000 mg/g) and followed for at least 5 years. The rate of change in estimated GFR (eGFR) adjusted for age and baseline eGFR was compared among ACR categories. RESULTS A higher baseline ACR predicted a faster decline in eGFR for both sexes. Even within the normal range (<30 mg/g), ACR > or =10 mg/g in women and > or =5 mg/g in men was associated with a significantly greater rate of decline in eGFR relative to subjects with ACR <5 mg/g. CONCLUSIONS Elevated ACR, even within the normal range, is associated with a faster decline in eGFR in diabetic patients.

Is a reduced estimated glomerular filtration rate a risk factor for stroke in patients with type 2 diabetes?

Although chronic kidney disease is a risk factor for cardiovascular disease it is unclear whether diabetic patients with a reduced glomerular filtration rate (GFR), independent of (micro)albuminuria, carry an increased risk of stroke. We therefore investigated the independent effect of estimated GFR (eGFR) on stroke events in patients with type 2 diabetes mellitus (T2DM). We studied T2DM patients with an eGFR >or=15 ml min(-1) per 1.73 m(2), who had no history of stroke. Patients were divided into four categories by the eGFR at baseline for comparison: >or=90, 60-89, 30-59 and 15-29 ml min(-1) per 1.73 m(2). The end point was an incident stroke event. The Cox proportional hazard model was used to calculate the hazard ratio (HR) and 95% confidence interval (CI). The study included a total of 1300 T2DM patients (546 women and 754 men) with a mean (+/-s.d.) age of 63+/-13 years. During a mean follow-up period of 3.7+/-1.4 years, 91 patients experienced an incident stroke event. Although a lower eGFR was associated with an increased stroke risk using a univariate model, statistical significance disappeared after adjusting for other risk factors including albuminuria. The HR (95% CI) was 0.75 (0.40-1.41, P=0.373), 0.99 (0.50-1.95, P=0.964) and 0.91 (0.36-2.28, P=0.844) for patients with eGFRs of 60-89, 30-59 and 15-29 ml min(-1) per 1.73 m(2), respectively, compared with patients with an eGFR >or=90. Clinical albuminuria remained a significant risk factor for stroke, and the adjusted HR compared with normoalbuminuria was 2.40 (1.46-3.95, P=0.001). In conclusion, the association between reduced GFR and stroke events in patients with T2DM is likely to be mediated by albuminuria.

Incorporation and translocation of 2-deoxy-2-[(18)F]fluoro-D-glucose in Sorghum bicolor (L.) Moench monitored using a planar positron imaging system.

[(18)F]FDG (2-deoxy-2-[(18)F]fluoro-D-glucose) was fed to a sorghum plant [Sorghum bicolor (L.) Moench] from the tip of a leaf and its movement was monitored using a planar positron imaging system (PPIS). [(18)F]FDG was uptaken from the leaf tip and it was translocated to the basal part of the shoots from where it moved to the roots, the tillers and the sheaths. Autoradiographic analysis of the distribution of (18)F, [(18)F]FDG and/or its metabolites showed translocation to the roots, tillers, and to the leaves that were younger than the supplied leaf. Strong labelling was observed in the basal part of the shoots, in the sheaths, the youngest leaf and the root tips. Our results indicate that [(18)F]FDG and/or its metabolites were absorbed from the leaf and translocated to the sites where nutrients are required. This strongly suggests that [(18)F]FDG can be utilised as a tracer to study photoassimilate translocation in the living plant. This is the first report on the use of [(18)F]FDG, which is routinely used as a probe for clinical diagnosis, to study source to sink translocation of metabolites in whole plants in real time.

Participation of the inositol phospholipid signaling pathway in the increase in cytosolic calcium induced by tributyltin chloride intoxication of chlorophyllous protozoa Euglena gracilis Z and its achlorophyllous mutant SM-ZK.

Exposure of tri-n-butyl tin chloride (TBTCl) as a stressor to Euglena gracilis Z causes rapid alteration of cell morphology followed by deflagellation. The present study was undertaken to reveal the mechanism of the cell response at a molecular level. Chlamydomonas reinhardtii, in this study E. gracilis Z and its achlorophyllous mutant SM-ZK, gave similar results when subject to the same stressor. Indeed, similar results were obtained with both strains. Next, assuming that the morphological alteration caused by TBTCl is mediated by the inositide phosphate-lipid signaling pathway, the effects of signal transduction and Ca2+ release reagents (mastoparan as a G-protein activator, neomycin as a phospholipase C inhibitor, verapamil as a Ca2+ channel blocker, and A23187 as a Ca2+ ionophore) on morphology and intracellular Ca2+ levels were examined with or without TBTCl. The data strongly suggest that the morphological alteration is mediated by an increase in Ca2+ linked to the inositol phosphatide pathway. The cellular response to signal transduction inducing reagents was compared between the E. gracilis chlorophyllous Z strain and its achlorophyllous mutant SM-ZK strain. Significant differences were observed between the Z and SM-ZK strains in terms of the stress response and intracellular Ca2+ level.

Long-term effects of subcutaneously injected 2,3,7,8-tetrachlorodibenzo-p-dioxin on the liver of rhesus monkeys.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accumulates and remains stable in the fatty tissues and liver of rodents for a long time. Considering the pronounced difference between species, long-term, low dose hepatic effects of TCDD were investigated after subcutaneous administration of TCDD into rhesus monkeys during pregnancy. Macroscopic and histopathological examination of the liver carried out 4 y after TCDD administration demonstrated intrahepatic focal fatty changes, infarction, hemorrhage, microthrombi-formation, sinusoidal ectasia, small hepatocyte hyperplasia, and increased number of alpha-smooth muscle actin (alpha-SMA)-positive cells. An electron microscopic study disclosed sinusoidal endothelial cell degeneration and injury in the liver of TCDD-treated monkeys. Western blot analysis showed downregulation of aryl hydrocarbon receptor (AhR) protein expression and decreased level of vascular endothelial (VE) cadherin but increased expression levels of CYP1A1 and transforming growth factor beta (TGF-beta) protein in the liver tissues. These changes observed in TCDD-exposed monkeys indicated sinusoidal endothelial cell injury and impairment in intrasinusoidal microcirculation. Infarction, focal fatty change, and microthrombi-formation are considered to be closely associated with intrahepatic circulatory impairment. Increased number of alpha-SMA-positive cells and decreased level of VE cadherin expression in the liver tissues might also be associated with sinusoidal endothelial cell injury. In addition, downregulation of AhR expression and increased CYP1A1 protein levels in the liver were consistent with persistent effects of TCDD. Although it has been reported that TCDD induced endothelial cell injury, this is the first report to describe vascular disorders and protein expression in the liver after injection with TCDD in a primate model.

Values of water-soluble vitamins in blood and urine of Japanese young men and women consuming a semi-purified diet based on the Japanese Dietary Reference Intakes.

We investigated the levels of water-soluble vitamins except for vitamin B6 in the blood and urine of Japanese college male (n = 10) and female (n = 10) students. They consumed for 7 d a semi-purified diet based on Japanese Dietary Reference Intakes to assess the Recommended Dietary Allowances (RDA) for water-soluble vitamins and to present some new normal values for blood and urine levels of water-soluble vitamins in Japanese. The blood and the 24-h urine samples were collected on the last day of the experiment and measured. The values of total vitamin B1 in whole blood, total vitamin B2 in whole blood, total cyanocobalamin in serum, total nicotinamide in whole blood, total pantothenic acid in whole blood, total folates in serum, total biotin in serum, and ascorbic acid in plasma were 104+/-17 pmol/mL (mean+/-SD), 216+/-25 pmol/mL, 0.34+/-0.05 pmol/mL, 59.1+/- 5.0 nmol/ mL, 2.45+/-0.37 nmol/mL, 15.6+/-4.6 pmol/mL, 8.3+/-0.5 pmol/mL, and 62+/-10 nmol/mL, respectively, in males, and 90+/-23, 234+/-18, 0.67+/-0.20, 61.9+/-6.0, 2.48+/-0.30, 30.2+/-8.6, 8.4+/-0.3, and 67+/-14, respectively, in females. There was a significant difference in the values of cyanocobalamin and total folates between men and women. The urinary excretion of vitamin B1, vitamin B2, cyanocobalamin, sum of the catabolic metabolites of nicotinamide, pantothenic acid, folates, biotin, and ascorbic acid were 665+/-114 nmol/d, 562+/-325 nmol/d, 93+/-31 pmol/d, 84+/-26 micromol/d, 9.3+/-2.3 micromol/d, 19.4+/-2.8 nmol/d, 83+/-18 pmol/d, and 148+/-51 micromol/d, respectively, in males, and 495+/-212, 580+/-146, 145+/-49, 83+/-19, 16.9+/-1.3, 22.7+/-2.7, 83+/-23, and 140+/-51, respectively, in females. There was a significant difference in the urinary excretion of cyanocobalamin, pantothenic acid and total folates between men and women. These values will be useful for the nutritional assessment of water-soluble vitamins for Japanese, although the examination period was short. In future, an experiment with various age groups and re-evaluation by repeated experiments will provide more accurate values.

[Metabolic disturbance of tryptophan-nicotinamide conversion pathway by putative endocrine disruptors, bisphenol A and styrene monomer].

Bisphenol A, a monomer of polycarbonate plastics, disturbed the conversion pathway of the amino acid tryptophan to the vitamin nicotinamide. The conversion ratio of tryptophan to nicotinamide was reduced to 1/15 by feeding a diet containing 1% bisphenol A. A putative disturbing reaction is kynurenine-->3-hydroxykynurenine, which is catalyzed by kynurenine monohydroxylase. This is an FAD-enzyme and requires NADPH as a coenzyme. Styrene monomer (1% addition to a normal diet) did not affect the food intake or the body weight, but slightly reduced the conversion ratio of tryptophan-nicotinamide.

Effects of dietary di(2-ethylhexyl)phthalate, a putative endocrine disrupter, on enzyme activities involved in the metabolism of tryptophan to niacin in rats.

We have reported that the conversion ratio of tryptophan to niacin increased with increasing dietary concentration of di(2-ethylhexyl)phthalate (DEHP); the conversion ratio was about 2.0% in the control rat, which increased by about 30% in the rat fed with 3.0% DEHP diet. In this study, we investigated whether this abnormal increase in the conversion ratio by DEHP occurred through the alteration of the enzyme activities involved in the metabolism of tryptophan to niacin. Rats were fed with a diet containing 0%, 0.1%, 0.5%, or 1.0% DEHP for 21 days. The nine kinds of enzyme activities involved in the biosynthesis and catabolism in the liver and kidney were measured. Based on previous findings that the formation of quinolinic acid and its' metabolites significantly increased with DEHP administration, we proposed that the activity of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase would be inhibited by DEHP intake. However, we found that the activities in the liver and kidney did not decrease in the rat fed with DEHP-containing diet. We discuss the discrepancy between the metabolite results and the enzyme activities.

Effects of dietary di(2-ethylhexyl)phthalate on the metabolism of tryptophan to niacin in mice.

We have reported the effect of di(2-ethylhexyl)phthalate (DEHP) on the tryptophan (Trp)-niacin pathway in rats. To clarify the universal effect of DEHP on rodents, we studied whether DEHP also has an effect on Trp metabolism in mice. Mice were fed a niacin-free, 20% casein diet supplemented with DEHP for 21 days. Feeding with DEHP decreased the body weight gain and increased the liver weight in correlation with the dose level of DEHP. The administration of DEHP significantly increased the formation of quinolinic acid and the lower metabolites of the Trp-niacin pathway. The flux of niacin in the lower part of the Trp-niacin pathway in mice was enhanced by feeding with DEHP.

Conversion ratio of tryptophan to niacin in Japanese women fed a purified diet conforming to the Japanese Dietary Reference Intakes.

In order to establish the human requirements of niacin, it is first important to know how much tryptophan is converted to niacin in the human body. In a general, 60 mg of tryptophan is equivalent to 1 mg of niacin, whereas the conversion ratio of tryptophan to niacin is yet to be confirmed. The aim of this study was to know the conversion ratio of tryptophan to niacin in Japanese females fed a purified diet, which followed the Japanese Dietary Reference Intakes. Ten young Japanese females were housed in the same facility and given the same daily living activity schedule for 7 d. The composition of their purified diet was conformed to the Dietary Reference Intakes in Japan. The diet was niacin free. In order to investigate the conversion ratio, daily urinary outputs were collected. Tryptophan-niacin metabolites in the urine were measured and the conversion ratio of tryptophan to niacin calculated. The conversion ratio was calculated by comparing the dietary intake of tryptophan and the sum of the niacin catabolites such as N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, which were derived only from the dietary intake of tryptophan. The ratio was calculated as 1.5 +/- 0.1 (mean +/-SE for 10 women; in molar basis) on the last day of the experiment. It was calculated that if the excretory percentage of niacin metabolites in the urine were 60%, of the tryptophan ingested, the conversion factor would be a value of 67, meaning that is 67 mg of tryptophan is equal to 1 mg of niacin.

Changes in the urinary excretion of the metabolites of the tryptophan-niacin pathway during pregnancy in Japanese women and rats.

NAD is biosynthesized from tryptophan. Therefore, experimental studies including tryptophan metabolism studies could provide insight into niacin nutrition in pregnancy. Our aim was to determine the change in niacin metabolism during pregnancy by a systemic investigation of how pregnancy alters the tryptophan-niacin metabolism in Japanese women and rats. For the human study, spot urine samples were collected from a total of 434 pregnant Japanese women who were at 5-40 wk of gestation, 50 women at 4-6 wk postpartum, and 10 nonpregnant women as the controls. For the animal study, pregnant rats were fed with a niacin-free diet, and daily urine samples were collected from day 6 of gestation to day 6 postpartum. The intermediates and metabolites of the tryptophan-niacin pathway in the urine samples were measured. The urinary excretions of niacin metabolites in humans and rats increased from mid pregnancy in a time-dependent manner, reached a peak of 2-3-fold during late pregnancy, and declined to control levels after childbirth.

Oxidative stress-induced cellular damage caused by UV and methyl viologen in Euglena gracilis and its suppression with rutin.

The effects of ultraviolet radiation (UV-A: 320-400 nm and UV-B: 280-320 nm) and methyl viologen (MV) single or combined exposure, on the cell growth, viability and morphology of two strains of the unicellular flagellate Euglena gracilis, using the Z strain as a plant model and the achlorophyllous mutant SMZ strain as an animal model were investigated. Cell growth was not affected by MV only, whereas UV-A or UV-B single and combined exposure with MV inhibited the cell growth or decreased the viability. The SMZ strain had a higher number of abnormal cells than the Z strain after the third dose of UV-B was delivered simultaneously with MV. The abnormal cell number decreased when E. gracilis SMZ cells were preincubated with 100 microM rutin prior to the UV-B and MV exposure. There were higher abnormal cell numbers with groups exposed to UV rather than MV single exposure. Combined exposure to UV-B and 200 microM MV induced the highest levels of TBARS in both strains, and with the supplementation of rutin these high levels were suppressed. These results suggest that UV-A or UV-B irradiation alone or combined with MV cause considerable oxidative damage in E. gracilis cells, and rutin supplementation may suppress their adverse effects.

Detoxifying effect of desalinated deep seawater on organotin-poisoned Euglena gracilis.

A Euglena gracilis Z strain was used as a biomarker to examine the detoxifying effects of desalinated deep seawater (DDSW) and surface seawater (DSSW) on the hazardous chemical, tributyltin chloride (TBTCl). A distinct restoration effect on cell motility was observed after incubating with DDSW. The effect was largely attributed to magnesium, calcium and silicon that are involved in cell motility and morphology.

Inactivation of staphylococcal enterotoxin-A with an electrolyzed anodic solution.

Electrolyzed anodic NaCl solutions [EW+], prepared by the electrolysis of 0.1% NaCl, have been shown to instantly inactivate most pathogens that cause food-borne disease. Elimination of food-borne pathogens does not necessarily guarantee food safety because enterotoxins produced by pathogens may remain active. We have tested whether EW+ can inactivate Staphylococcal enterotoxin A (SEA), one of the major enterotoxins responsible for food poisoning. Fixed quantities of SEA were mixed with increasing molar ratios of EW+, and SEA was evaluated by reversed-phase passive latex agglutination (RPLA) test, immunoassay, native polyacrylamide gel electrophoresis (PAGE), and amino acid analysis after 30 min incubations. Exposure of 70 ng, or 2.6 pmol, of SEA in 25 microL of PBS to a 10-fold volume of EW+, or ca. 64.6 x 10(3)-fold molar excess of HOCl in EW+, caused a loss of immuno-reactivity between SEA and a specific anti-SEA antibody. Native PAGE indicated that EW+ caused fragmentation of SEA, and amino acid analysis indicated a loss in amino acid content, in particular Met, Tyr, Ile, Asn, and Asp. Staphylococcal enterotoxin-A excreted into culture broth was also inactivated by exposure to an excess molar ratio of EW+. Thus, EW+ may be a useful management tool to ensure food hygiene by food processing industries.

Decontamination of aflatoxin-forming fungus and elimination of aflatoxin mutagenicity with electrolyzed NaCl anode solution.

Electrolysis of a 0.1% (17.1 mM) solution of NaCl using separate anode and cathode compartments gives rise to solutions containing active chemical species. The strongly acidic "anode solution" (EW+) has high levels of dissolved oxygen and available chlorine in a form of hypochlorous acid (HOCl) with a strong potential for sterilization, which we have investigated here. Exposing Aspergillus parasiticus at an initial density of 10(3)spores in 10 microL to a 50-fold volume (500 microL) of EW+ containing ca. 390 micromol HOCl for 15 min at room temperature resulted in a complete inhibition of fungal growth, whereas the cathode solution (EW-) had negligible inhibitory effects. Moreover, the mutagenicity of aflatoxin B(1) (AFB(1)) for Salmonella typhimurium TA-98 and TA-100 strains was strongly reduced after AFB(1) exposure to the EW+ but not with the EW-. In high-performance liquid chromatography analysis, the peak corresponding to AFB(1) disappeared after treatment with the EW+, indicating decomposition of the aflatoxin. In contrast, the routinely used disinfectant sodium hypochlorite, NaOCl, of the same available chlorine content as that of EW+ but in a different chemical form, hypochlorite (OCl-) ion, did not decompose AFB(1) at pH 11. However, NaOCl did decompose AFB(1) at pH 3, which indicated that the principle chemical formula to participate in the decomposition of AFB(1) is not the OCl- ion but HOCl. Furthermore, because the decomposition of AFB(1) was suppressed by pretreating the EW+ with the OH radical scavenger thiourea, the chemical species responsible for the AFB(1)-decomposing property of the EW+ should be at least due to the OH radical originated from HOCl. The OH in EW+ was proved by electron spin resonance analysis.