Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide.

AIMS: To develop a quick and accurate PCR-based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. METHODS AND RESULTS: The number of BbrY in faeces was detected by using strain-specific quantitative real-time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA-intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10(5) -10(9) cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (± 1·5) log CFU g(-1) [mean (± SD)] of BbrY was detected in faeces by using strain-specific transgalactosylated oligosaccharide-carbenicillin (T-CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (± 1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (± 0·8) log cells per g. CONCLUSIONS: Strain-specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. SIGNIFICANCE AND IMPACT OF THE STUDY: Combination of strain-specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.

Health issues today in affected areas near Fukushima Daiichi nuclear power plant.

Due to vigorous efforts to decontaminate the environment following the accident at Fukushima Daiichi nuclear power plant, the size of the difficult-to-return zone has reduced significantly and people have started returning to their homes. As the population has increased, medical needs have ensued. A marked increase in traffic as well as decontamination and reconstruction projects has led to an increase in the number of road traffic and occupational accidents. Acceleration of population aging has resulted in an increased number of elderly residents with multiple medical problems. Uncontrolled/untreated medical problems among middle-aged to older workers have made them susceptible to deterioration of health conditions. Insufficient social support for elderly people living alone has resulted in delayed access to medical care. Early intervention and the prevention of health deterioration are instrumental. When responding to medical needs, proactive approaches, including home visits for elderly patients and health promotion, have been implemented. Human resource development is crucial to ensure the sustainability of these activities.

Development of an application tool to support returnees in Fukushima.

To promote radiation protection and health promotion among returning residents (returnees) in coastal areas of Fukushima, eHealth principles were used to develop a new application tool (app) that can record radiation exposure and health status while providing comprehensive support to returnees. Intended users are returnees and health and welfare workers. After assessing their needs, a flowchart and prototype for operational logic were created using commercially available software tools. Professional developers will focus on improving the user interface and ensuring data security. The finished app will be compatible with mobile telephones and tablets. Utility and ease of use are paramount to serve returnees of all ages effectively.

Tryptophan aspartate-containing coat protein (CORO1A) suppresses Toll-like receptor signalling in Mycobacterium leprae infection.

Mycobacterium leprae is an intracellular pathogen that survives within the phagosome of host macrophages. Several host factors are involved in producing tolerance, while others are responsible for killing the mycobacterium. Tryptophan aspartate-containing coat protein (TACO; also known as CORO1A or coronin-1) inhibits the phagosome maturation that allows intracellular parasitization. In addition, the Toll-like receptor (TLR) activates the innate immune response. Both CORO1A and TLR-2 co-localize on the phagosomal membrane in the dermal lesions of patients with lepromatous leprosy. Therefore, we hypothesized that CORO1A and TLR-2 might interact functionally. This hypothesis was tested by investigating the effect of CORO1A in TLR-2-mediated signalling and, inversely, the effect of TLR-2-mediated signalling on CORO1A expression. We found that CORO1A suppresses TLR-mediated signal activation in human macrophages, and that TLR2-mediated activation of the innate immune response resulted in suppression of CORO1A expression. However, M. leprae infection inhibited the TLR-2-mediated CORO1A suppression and nuclear factor-kappaB activation. These results suggest that the balance between TLR-2-mediated signalling and CORO1A expression will be key in determining the fate of M. leprae following infection.

Circulating KL-6/MUC1 as an independent predictor for disseminated intravascular coagulation in acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome (ARDS) patients show high levels of circulating mucin including KL-6/MUC1 (soluble MUC1 mucin). Because cancer mucin can bind vascular endothelial cells and platelets via selectins, mucin-selectin interactions are reported to trigger platelet aggregation and intravascular coagulation. Therefore, we hypothesized that KL-6/MUC1 is involved in the pathogenesis of disseminated intravascular coagulation (DIC) in ARDS. The aim of the current study is to evaluate the association between circulating KL-6/MUC1 and DIC in ARDS patients. DESIGN: Observational study with structured follow-up. SETTING: Intensive care unit in Hiroshima University Hospital. SUBJECTS: Fifty-six newly diagnosed patients with ARDS. INTERVENTIONS: Circulating levels of KL-6/MUC1 were measured during diagnosis and serially measured during the clinical course along with indices of respiratory failure, inflammation, coagulation and fibrinolysis and multiple organ dysfunction. RESULTS: Acute respiratory distress syndrome patients complicated with DIC showed significantly higher levels of serum KL-6/MUC1 than patients without DIC during the clinical course. Amongst the parameters analysed at diagnosis of ARDS, KL-6/MUC1 was an independent predictor for DIC complication. The baseline level of circulating KL-6/MUC1 at diagnosis of ARDS was significantly correlated with an increased DIC score following ARDS diagnosis. Using an optimum cutoff level of KL-6/MUC1 obtained by a receiver operating characteristic curve, the sensitivity and specificity for predicting future DIC development in ARDS patients were 88.9% and 55.3%, respectively. CONCLUSIONS: These results suggest that KL-6/MUC1 is associated with DIC development in ARDS patients. Elevated levels of KL-6/MUC1 at diagnosis could be a predictor of DIC complication in ARDS.

Medical and health surveillance in postaccident recovery: experience after Fukushima.

The accident at Fukushima Daiichi nuclear power plant occurred following the huge tsunami and earthquake of 11 March 2011. After the accident, there was considerable uncertainty and concern about the health effects of radiation. In this difficult situation, emergency responses, including large-scale evacuation, were implemented. The Fukushima Health Management Survey (FHMS) was initiated 3 months after the accident. The primary purposes of FHMS were to monitor the long-term health of residents, promote their well-being, and monitor any health effects related to long-term, low-dose radiation exposure. Despite the severity of the Fukushima accident and the huge impact of the natural disaster, radiation exposure of the public was very low. However, there were other serious health problems, including deaths during evacuation, increased mortality among displaced elderly people, mental health and lifestyle-related health problems, and social issues after the accident. The Nuclear Emergency Situations - Improvement of Medical and Health Surveillance (SHAMISEN) project, funded by the Open Project For European Radiation Research Area, aimed to develop recommendations for medical and health surveillance of populations affected by previous and future radiation accidents. This paper briefly introduces the points that have been learned from the Fukushima accident from the perspective of SHAMISEN recommendations.

Emergency Responses and Health Consequences after the Fukushima Accident; Evacuation and Relocation.

The Fukushima accident was a compounding disaster following the strong earthquake and huge tsunami. The direct health effects of radiation were relatively well controlled considering the severity of the accident, not only among emergency workers but also residents. Other serious health issues include deaths during evacuation, collapse of the radiation emergency medical system, increased mortality among displaced elderly people and public healthcare issues in Fukushima residents. The Fukushima mental health and lifestyle survey disclosed that the Fukushima accident caused severe psychological distress in the residents from evacuation zones. In addition to psychiatric and mental health problems, there are lifestyle-related problems such as an increase proportion of those overweight, an increased prevalence of hypertension, diabetes mellitus and dyslipidaemia and changes in health-related behaviours among evacuees; all of which may lead to an increased cardiovascular disease risk in the future. The effects of a major nuclear accident on societies are diverse and enduring. The countermeasures should include disaster management, long-term general public health services, mental and psychological care, behavioural and societal support, in addition to efforts to mitigate the health effects attributable to radiation.

Skeletal malformations in rat offspring. Long-term effect of maternal insulin-induced hypoglycemia during organogenesis.

We studied the effect of maternal hypoglycemia on skeletal growth in the offspring of nondiabetic and diabetic rats. Female Wistar rats were injected with streptozocin (30 mg/kg i.v.) 2-3 wk before mating, and diabetes was confirmed by an intraperitoneal glucose tolerance test. On postconception day 9.5 or 10.5, both control and diabetic dams received saline or Actrapid human insulin (400 mU/rat i.p.). Hypoglycemia (approximately 2.8 mM) was induced for 120 min in the insulin-treated mothers. Pregnancy was terminated on gestational day 20. Fetal bones and cartilage were double-stained with alizarin red S and alcian blue 8GS. Insulin-induced hypoglycemia caused delayed ossification in the fetuses of the control dams. The number of malformations, e.g., costal fusion waves, increased greatly. These effects were more striking in the fetuses of dams that had received insulin on day 10.5 rather than on day 9.5 of embryo development. This type of insulin-induced hypoglycemia further delayed ossification of the fetal bones in diabetic dams. The influence of maternal hypoglycemia on skeletal malformations and/or variations was greater in the fetuses of diabetic dams than in the fetuses of control dams. These data suggest that maternal hypoglycemia in early pregnancy has a striking effect on skeletal growth and malformations in fetuses. In addition, mild glucose intolerance in dams may amplify these hypoglycemic effects.

Somatostatin-like immunoreactivity in human peripheral plasma measured by radioimmunoassay following affinity chromatography.

Somatostatin-like immunoreactivity (SLI) concentrations were determined in human peripheral plasma using affinity chromatography followed by radioimmunoassay. In normal subjects, fasting SLI ranged from 2.9 to 22.0 pg/ml with a mean +/- SE value of 10.2 +/- 2.1 pg/ml. In totally pancreatectomized or gastrectomized patients, fasting SLI levels were not different from the values in normal subjects. In patients with medullary thyroid carcinoma, fasting SLI ranged from 11.8 to 71.0 pg/ml with a mean of 29.3 +/- 12.3 pg/ml, which was significantly higher than normal values (P less than 0.01). Following meal ingestion, plasma SLI increased significantly in normal subjects from a basal level of 9.1 +/- 2.1 pg/ml to a peak value of 15.4 +/- 2.9 pg/ml (P less than 0.02). These results indicate that radioimmunoassay combined with affinity chromatography provides an accurate method of measuring SLI in human plasma.

Starvation-induced changes of somatostatin, glucagon, and insulin secretion from the isolated perfused rat pancreas.

The effect of 16- and 48-h fasting on pancreatic somatostatin, insulin, and glucagon secretion was studied, using the isolated perfused rat pancreas. In the presence of 4.4 mM glucose, basal somatostatin and insulin concentrations in the perfusate were significantly lower in 48-h fasted rats than in fed animals, whereas basal glucagon secretion was significantly elevated in fasted rats. The infusion of 19 mM arginine significantly augmented secretion of somatostatin and glucagon and attenuated insulin secretion in 48-h fasted rats. It is concluded that fasting causes a decrease in basal pancreatic somatostatin secretion in vitro, although the response to arginine is rather exaggerated. Insulin and glucagon secretion also changed during the fasting. These results suggest that not only insulin and glucagon, but also somatostatin contribute to nutrient homeostasis.

[Mitral valve disease in elderly patients].

It seems that mitral valve surgery in patients older than 80 years increase against the background of the aging society. Basically, there are not particular differences between elderly patients and younger patients in procedures for diagnosis and operative indication in mitral valve diseases. However, cardiac surgery for patients older than 80 years has been reported to have high mortality rate. Therefore, it is necessary that we should evaluate and treat complications of important organs such as brain/coronary artery/lungs/liver/kidney, adequately. Our mortality rate of the whole mitral valve operations was 2.4% and that of patients older than 75 years was 7.9%. This result compares favorably with those reported from other institutions. However, when we perform mitral valve operations for patients older than 80 years, it is necessary to investigate activities of daily living and quality of life before and after the operation.

Rat liver postischemic lipid peroxidation and vasoconstriction depend on ischemia time.

In this investigation, we used chemiluminescence to study the ability of increasing durations of ischemia (1, 2, or 2.5 h) to induce enhanced generation of reactive oxygen species in a crystalloid perfused rat liver model. To evaluate the effect of reactive oxygen species generation upon the development of the postischemic hypoperfusion, hepatic vascular resistance was simultaneously monitored. One hour of ischemia did not produce sustained reactive oxygen species generation or development of no-reflow. Two hours of ischemia did not result in sustained reactive oxygen species generation but did produce no-reflow. Sustained reactive oxygen production was achieved after 2.5 h of ischemia and was accompanied by the development of no-reflow. We found that 2.5 h of ischemia is the threshold for sustained lipid peroxidation. Both lipid peroxidation and no-reflow could be mitigated through the administration of superoxide dismutase. Superoxide dismutase could reduce the amount of cell injury due to the enhanced lipid peroxidation induced by 2.5 h of ischemia. Limitation of reactive oxygen species generation to a critical threshold, either by restricting the duration of ischemia or by pharmacological intervention, may be an important means of preventing further cellular injury through no-reflow and lipid peroxidation.

Enhanced adjuvant effect of granulocyte-macrophage colony-stimulating factor plus interleukin-12 compared with either alone in vaccine-induced tumor immunity.

Using the poorly immunogenic D5 murine melanoma, we examined the adjuvant effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) secretion by gene-modified tumor cells inoculated as a vaccine to prime tumor-draining lymph nodes (TDLNs). D5 transfectants that secreted IL-12 or GM-CSF alone were compared with a double transfectant that secreted equivalent amounts of both cytokines. TDLN cells harvested 9-10 days after subcutaneous tumor inoculation were cultured sequentially in anti-CD3 and IL-2 and assessed for antitumor reactivity against wild-type D5 tumor. The double transfectant-induced TDLN effector cells had greater cytotoxicity in a long-term assay than TDLN cells primed by single transfectants. In adoptive immunotherapy, the TDLN cells primed by the double transfectant were significantly better at mediating the regression of established tumors compared with the TDLN cells elicited by the single transfectants. Both IL-12 and GM-CSF had adjuvant effects in promoting tumor-reactive TDLN cells, but the combination was better than either alone. These observations suggest that the immunomodulation roles of IL-12 and GM-CSF are different and complementary.

Novel point mutation in the uroporphyrinogen III synthase gene causes congenital erythropoietic porphyria of a Japanese family.

The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in a member of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Günther's disease. The patient was homozygous for a novel missense mutation: a G to T transition of nucleotide 7 that predicted a valine to phenylalanine substitution at residue 3 (V3F). The parents were heterozygous for the same mutation. The loss of UROIIIS activity was verified by an in vitro assay system. The corresponding mutated protein was expressed in Escherichia coli and no residual activity was observed. Further studies are needed to determine whether the mutations of the UROIIIS gene (UROS) have a specific profile in Japan compared to European or American countries.

Unique case of growth hormone (GH) deficiency accompanied by clinical anophthalmia, hypoplastic orbits, digital dysplasia, short stature, obesity, and diabetes mellitus.

A 43-year-old female was admitted to our hospital for polydipsia and hyperglycemia. She had total blindness and globes were not recognized by inspection, indicating clinical anophthalmia. Physical examination revealed short stature, obesity, prematurely gray hair, shortness of fingers and toes, syndactyly, and multiple dental caries. Laboratory examination showed hyperglycemia, increased glycosilated hemoglobin (HbA1c) and insulin resistance on euglycemic glucose clamp. Blunted growth hormone (GH) secretion was shown in response to insulin-induced hypoglycemia, arginine infusion, and GH-releasing hormone (GHRH) loading test, and in 24 h spontaneous GH profile. Magnetic resonance imaging (MRI) and computed tomography (CT) showed dysostosis of orbit, defect of optic nerve, enlarged suprasellar cistern, and prolonged pituitary stalk. This may be the first report of a unique case with GH deficiency accompanied by clinical anophthalmia, hypoplastic orbits, digital dysplasia, short stature, obesity, and diabetes mellitus.

Impairment of insulin-stimulated GLUT4 translocation in skeletal muscle and adipose tissue in the Tsumura Suzuki obese diabetic mouse: a new genetic animal model of type 2 diabetes.

BACKGROUND: In skeletal muscle and adipocytes, insulin-stimulated glucose transport has been known to occur through the translocation of glucose transporter (GLUT) 4 from the intracellular pool to the plasma membrane. The Tsumura Suzuki obese diabetic (TSOD) mouse, a new genetic animal model of type 2 diabetes, develops moderate degrees of obesity and diabetes that are especially apparent in animals more than 11 weeks old. A defect in insulin stimulation of GLUT4 translocation also contributes to the characteristics of type 2 diabetes. OBJECTIVE: To characterize this mouse further, we examined the alteration in insulin-stimulated GLUT4 translocation in the skeletal muscle and adipose tissue. METHODS: For glucose and insulin tolerance tests, the mice were given glucose or insulin and blood samples were collected. After isolation of low-density microsomal membrane and plasma membrane from skeletal muscle and adipose tissue, insulin-stimulated translocation of GLUT4 in these TSOD mice was examined by Western blot. RESULTS AND CONCLUSIONS: TSOD mice showed a significant increase in blood glucose after the glucose load, and exhibited a significantly attenuated decrease in blood glucose concentrations after administration of insulin, compared with that in control Tsumura Suzuki non-obese (TSNO) mice. The insulin-stimulated translocation of GLUT4 from low-density microsomal membranes to plasma membrane was significantly reduced in both skeletal muscle and adipose tissue of TSOD mice. These results indicate that the reduced insulin sensitivity in diabetic TSOD mice is presumably due, at least in part, to the impaired GLUT4 translocation by insulin in both skeletal muscle and adipocytes.

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets.

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.

Effects of additives on irreversible inactivation of lysozyme at neutral pH and 100 degrees C.

The mechanism of irreversible inactivation of lysozyme at neutral pH at 100 degrees C, and effects of additives on the inactivation were investigated. The thermoinactivation of lysozyme at neutral pH was caused by intra- and intermolecular disulfide exchange and the production of irreversibly denatured lysozyme, which was destabilized by multiple chemical reactions other than disulfide exchange. In addition, independently, deamidation slightly affected the inactivation by causing a decrease of electrostatic interaction between positive charges of lysozyme and negative charges of the bacterial cell wall. As for the effects of additives on the inactivation, a small amount of copper ion suppressed intra- and intermolecular disulfide exchange by catalyzing air oxidation of heat-induced trace amounts of free thiols, and organic reagents (acetamide, ethanol, and glycerol) changed the mechanism of the inactivation to that under acidic conditions by shifting the pKa values of dissociable residues and also suppressed intermolecular disulfide exchange by decreasing hydrophobic interactions.

Insulin secretion and biosynthesis by the perfused pancreas of spontaneously hypertensive rats.

Insulin secretory activity was compared in the spontaneously hypertensive rat (SHR) versus the normotensive Wistar-Kyoto (WKY) rat and Wistar rat. When the isolated pancreas was perfused with 16.7 mmol/L glucose, insulin release was significantly greater in the SHR versus the other groups. On the other hand, there was no difference in arginine (19 mmol/L)-induced insulin secretion among the three groups. To determine insulin biosynthesis during glucose stimulation, the pancreas was perfused with 16.7 mmol/L glucose for 180 minutes. Insulin secretion was greater in SHR versus WKY and Wistar rats, but the net increase in insulin content was not different between the three groups. These results strongly suggest that in vivo hyperinsulinemia in the SHR is associated with increased in vitro insulin secretion in response to glucose. The mechanisms by which enhanced glucose-induced insulin secretion is linked to hypertension in the SHR remain unclear.

Effect of pregnancy on insulin metabolism in spontaneously hypertensive rats.

Several lines of evidence suggest that insulin resistance and/or hyperinsulinemia may play an important role in the pathogenesis of hypertension. We studied the effect of pregnancy on insulin metabolism in spontaneously hypertensive rats (SHRs) and in Wistar-Kyoto rats (WKYs) as a control. Pregnancy markedly reduced blood pressure in both strains of rats, but insulin resistance as determined by the hyperinsulinemic glucose clamp (10 mU/kg/min) increased in SHRs and was unchanged in WKYs. The plasma insulin response to an intravenous glucose challenge in SHRs was low and did not change with pregnancy. Therefore, it is suggested that the regulation of blood pressure in these animals is linked to an unknown factor rather than to insulin resistance and hyperinsulinemia. Fetuses from SHRs had a lower body weight and plasma glucose level and higher plasma insulin and pancreatic insulin levels than those from WKYs. Thus, fetal hyperinsulinemia in the SHR may be linked to the development of hypertension in adulthood.