Genetics in pulmonary arterial hypertension in a large homogeneous Japanese population.

Pulmonary arterial hypertension (PAH) is a rare but serious disease with a grave prognosis. Bone morphogenetic protein type 2 receptor (BMPR2) gene is a strong pathogenic factor for PAH. As a collaborative team from Kyorin University and Keio University in Japan, we have analyzed the BMPR2 gene in 356 probands and more than 50 family members, including secondary patients. Importantly, the study population is a racially, ethnically, and socially homogeneous population. In PAH patients, there is a high incidence of unique mutations in BMPR2, and several mutations are frequently observed in the Japanese population, suggesting that these common and recurring mutations may be highly pathogenic or have high penetrance, explaining why they are found frequently throughout the world. We have also mapped each breakpoint of exonic deletions/duplications and found that most break and rejoining points are in the Alu elements. Reviewing the distribution of the reported mutations on each exon of BMPR2 revealed that the number and frequency of mutations are imbalanced among exons. The penetrance of BMPR2 gene mutations was 3-fold higher in females than males. Full elucidation of BMPR2-mediated pathogenic mechanisms in PAH requires persistent efforts to achieve precision or individualized medicine as a therapeutic strategy for PAH.

Growth of In x Ga1-x Sb alloy semiconductor at the International Space Station (ISS) and comparison with terrestrial experiments.

BACKGROUND: In x Ga1-x Sb is an important material that has tunable properties in the infrared (IR) region and is suitable for IR-device applications. Since the quality of crystals relies on growth conditions, the growth process of alloy semiconductors can be examined better under microgravity (µG) conditions where convection is suppressed. AIMS: To investigate the dissolution and growth process of In x Ga1-x Sb alloy semiconductors via a sandwiched structure of GaSb(seed)/InSb/GaSb(feed) under normal and µG conditions. METHODS: In x Ga1-x Sb crystals were grown at the International Space Station (ISS) under µG conditions, and a similar experiment was conducted under terrestrial conditions (1G) using the vertical gradient freezing (VGF) method. The grown crystals were cut along the growth direction and its growth properties were studied. The indium composition and growth rate of grown crystals were calculated. RESULTS: The shape of the growth interface was nearly flat under µG, whereas under 1G, it was highly concave with the initial seed interface being nearly flat and having facets at the peripheries. The quality of the µG crystals was better than that of the 1G samples, as the etch pit density was low in the µG sample. The growth rate was higher under µG compared with 1G. Moreover, the growth started at the peripheries under 1G, whereas it started throughout the seed interface under µG. CONCLUSIONS: Kinetics played a dominant role under 1G. The suppressed convection under µG affected the dissolution and growth process of the In x Ga1-x Sb alloy semiconductor.

Prevalence and growth kinetics of Shiga toxin-producing Escherichia coli (STEC) in bovine offal products in Japan.

Recent epidemiological data suggest a link between the consumption of bovine offal products and Shiga toxin-producing Escherichia coli (STEC) infection in Japan. This study thus examined the prevalence of STEC in various types of these foods. PCR screened 229 bovine offal products for the presence of Shiga toxin (stx) gene. Thirty-eight (16·6%) samples were stx positive, of which eight were positive for rfbE(O157) and three were positive for wzy(O26). Four O157 and one O26 STEC isolates were finally obtained from small-intestine and omasum products. Notably, homogenates of bovine intestinal products significantly reduced the extent of growth of O157 in the enrichment process compared to homogenates of beef carcass. As co-incubation of O157 with background microbiota complex from bovine intestinal products in buffered peptone water, in the absence of meat samples, tended to reduce the extent of growth of O157, we reasoned that certain microbiota present in offal products played a role. In support of this, inoculation of generic E. coli from bovine intestinal products into the homogenates significantly reduced the extent of growth of O157 in the homogenates of bovine intestinal and loin-beef products, and this effect was markedly increased when these homogenates were heat-treated prior to inoculation. Together, this report provides first evidence of the prevalence of STEC in a variety of bovine offal products in Japan. The prevalence data herein may be useful for risk assessment of those products as a potential source of human STEC infection beyond the epidemiological background. The growth characteristic of STEC O157 in offal products also indicates the importance of being aware when to test these food products.

Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota.

AIMS: To develop species-specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by fluorescence whole-cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides-Parabacteroides microbiota of conventional mice and specific pathogen-free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group-1, Group-2 and Group-3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides-Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. CONCLUSIONS: Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides-Parabacteroides microbiota. The Group-3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group-1 and Group-2. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides-Parabacteroides community.

Antihypertensive effect of nuatigenin-3-O-ß-chacotriose from Solanum sisymbriifolium Lam. (Solanaceae) (ñuatî pytâ) in experimentally hypertensive (ARH+DOCA) rats under chronic administration.

The aim of the study is to assess the hypotensive properties of the hydro-ethanolic crude root extract (CRE), the n-butanol fraction (F(BtOH)) and nuatigenin-3-O-ß-chacotriose, from Solanum sisymbriifolium Lam., in adrenal regeneration hypertension+deoxycorticosterone acetate (ARH+DOCA) rats, following a chronic administration. The roots of S. sisymbriifolium Lam. (Solanaceae) were extracted by reflux with ethanol-water 7:3 and the active extract was fractionated by bioassay-guided liquid-liquid separation. Nuatigenin-3-O-ß-chacotriose (B(3-1)) was identified as the main hypotensive compound from the crude drug by spectroscopic methods. Immature Wistar rats of both sexes were submitted to both surgery and deoxycorticosterone acetate treatment to obtain adrenal regeneration hypertensive rats (ARH+DOCA). Different groups of experimentally induced hypertensive rats were randomly allotted and received during 16 weeks a daily oral administration of 1% saline solution (0.1 mL/100g body weigh), 100.0 mg/kg of CRE, 10.0, 30.0 and 50.0 mg/kg of F(BtOH), and 1.0 mg/kg of B(3-1), respectively. In addition, two groups of ARH+DOCA rats were randomly assigned to receive either B(3-1) (1.0 mg/kg/day) or 1% of saline solution (0.1 mL/100g body weight/day) for 7 weeks and then a cross over procedure was performed in order to complete the 16th-week treatment. After 16 weeks of oral administration of crude root extract (CRE), butanolic fraction (F(BtOH)) and nuatigenin-3-O-ß-chacotriose (B(3-1)) a significant reduction of blood pressure value was induced in hypertensive animals (ARH+DOCA) in comparison to the control group receiving 1% saline solution, at the end of experiment. Administration of B(3-1) (1.0 mg/kg/day p.o.) to ARH+DOCA rats provoked a significant reduction of blood pressure, observed gradually from 5th week (p<0.05) to the end of the 16th week period of treatment (p<0.01). Moreover, in a cross over design it was observed that the reduction of blood pressure to normotensive condition is associated to B(3-1). The latest demonstrated that the blood pressure-lowering effect, in clearly hypertensive animals, is reversible and depend upon administration of nuatigenin-3-O-ß-chacotriose (B(3-1)). Our results demonstrated that daily oral administration of CRE, F(BtOH) and nuatigenin-3-O-ß-chacotriose from S. sisymbriifolium for a 16-week period exhibits an antihypertensive effect in experimentally hypertensive (ARH+DOCA) rats.

Coffee consumption but not green tea consumption is associated with adiponectin levels in Japanese males.

PURPOSE: Coffee is among the most widely consumed beverages in the world. Numerous epidemiological studies have reported a significant inverse association between coffee consumption and risk of type 2 diabetes mellitus, but the underlying mechanisms are still not fully understood. Therefore, we conducted an epidemiological study to clarify the relationship between coffee consumption and adiponectin levels in Japanese males. We also evaluated whether green tea consumption affected adiponectin levels. METHODS: We carried out a cross-sectional study. The subjects were 665 male employees in Japan. Coffee consumption was assessed, using a self-administered questionnaire, as the number of times per week and cups per day respondents drank, and subjects were grouped into four levels (non, 1-5 times/week, 1-2 cups/day and ≥3 cups/day). RESULTS: The means of adiponectin levels were positively associated with coffee consumption. A dose-response relationship was found between coffee consumption and circulating adiponectin levels. The relationship remained significant after adjustment for potential confounding factors (P for trend <0.05). However, green tea consumption was not significantly associated with adiponectin levels (P for trend = 0.90). CONCLUSIONS: We not only revealed that habitual coffee consumption is associated with higher adiponectin levels in Japanese males but also found a dose-dependent association between coffee consumption and adiponectin levels. Therefore, our study suggested that coffee components might play an important role in the elevation of adiponectin level.

Evaluating measles surveillance: comparison of sentinel surveillance, mandatory notification, and data from health insurance claims.

Inadequate notification is a recognized problem of measles surveillance systems in many countries, and it should be monitored using multiple data sources. We compared data from three different surveillance sources in 2007: (1) the sentinel surveillance system mandated by the Act on Prevention of Infectious Diseases and Medical Care for Patients Suffering Infectious Diseases, (2) the mandatory notification system run by the Aichi prefectural government, and (3) health insurance claims (HICs) submitted to corporate health insurance societies. For each dataset, we examined the number of measles cases by month, within multiple age groups, and in two categories of diagnostic test groups. We found that the sentinel surveillance system underestimated the number of adult measles cases. We also found that HIC data, rather than mandatory notification data, were more likely to come from individuals who had undergone laboratory tests to confirm their measles diagnosis. Thus, HIC data may provide a supplementary and readily available measles surveillance data source.

16S rRNA gene sequence-based analysis of clostridia related to conversion of germfree mice to the normal state.

AIMS: To determine phylogenetic groups of clostridia inhabiting the mouse intestine that are essential for normalization of germfree (GF) mice. METHODS AND RESULTS: Using both the culture method and cloning, clostridia inhabiting the mouse intestine were isolated, and phylogenetic analysis based on 16S rRNA gene sequences was carried out. As a result, the isolates were found to have novel sequences, and no isolate was determined to be identical to previously known identified clostridia. Although the taxonomy of mouse intestinal clostridia was complex, many of them belonged to Clostridium clusters XIVa and IV in conventional (CV) and limited flora mice and ex-germfree mice administered chloroform-treated CV mouse faeces. The clostridia that belonged to cluster XIVa were most often present and showed the highest diversity. CONCLUSIONS: Clostridia belonging clusters XIVa and IV are dominant in the mouse intestine as in other gut ecosystems. The novel groups in these clusters are essential for normalization of GF mice. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be applied in the strict control of mouse intestinal microbiota and will provide important information for normalization of GF mice and also for research on microbiology of the mouse intestine.

Is heat shock protein 60 associated with type 2 diabetes mellitus?

AIMS: HSP60 plays a protective role against heat, oxidative injury and ultraviolet. Recently, animal and clinical studies have suggested that HSP60 plays a role in various diseases. However, few epidemiological studies have demonstrated an association between HSP60 levels and type 2 diabetes mellitus. Therefore, an epidemiological study was conducted to examine the association of HSP60 with type 2 diabetes mellitus. METHODS: This study included 83 type 2 diabetes mellitus patients and 161 controls that were recruited from male employees who received annual health check-ups between 2005 and 2007. The serum HSP60 levels were measured using the ELISA method. RESULTS: Because the HSP60 levels were not detectable (<3.125 ng/mL) in 48.0% of the study subjects, HSP60 levels were divided into two categories (detectable or undetectable). A logistic regression analysis showed that the subjects in the undetectable had a 2.03 times higher risk of diabetes mellitus than those in the detectable after adjustment for age, BMI and rate of hypertension medication. CONCLUSIONS: This study was the first epidemiological study to demonstrate an association between type 2 diabetes mellitus and HSP60, thus suggesting that HSP60 may play an important role in the type 2 diabetes mellitus pathology.

Haematological data for Matsumoto Eosinophilic Shinshu rats as determined by an automated haematology analyser.

The Matsumoto Eosinophilic Shinshu (MES) rat originated from an inbred mutant colony of rats with spontaneous eosinophilia. As part of an investigation of the pathogenesis of the MES rat, we examined the haematology data for 106 males and 88 females and age-associated changes using an automated haematology analyser, flow cytometric analysis and morphological examination. The data at 10 weeks of age showed the MES rats had higher counts for eosinophils and neutrophils, slightly higher counts for lymphocytes, monocytes, basophils, and large unstained cells (LUCs), and slightly lower values for the erythrocytic parameters when compared with Sprague-Dawley (SD) rats. In data for MES rats aged 8 to 20 weeks, eosinophil counts increased with age up to 20 weeks together with some increased neutrophil counts. After 11 weeks of age, counts for lymphocytes, monocytes, basophils, and LUCs in the MES rats were also slightly increased. In female MES rats, flow cytometric analysis showed increased counts for pan-T+ cells, but blasts, abnormal granulocytes and lymphocytes were not detected morphologically. The MES rat characterized by the haematological findings could be a useful animal model for studies of hypereosinophilia.

A yeast DNA microarray for the evaluation of toxicity in environmental water containing burned ash.

Numerous studies on the hazard assessment and epidemiological health responses to burned ash have been reported. However, there is little information on the potential toxicity of unknown chemical complexes in burned ash. For an overall evaluation of the multiple toxicities of burned ash, a DNA microarray was used in this study, as a new attempt to assess these toxicities. Using the global gene expression on yeast DNA chip to reflect the changes in mRNA levels, our study discovered a lot of evidences for the action of cell homeostasis and stress response etc., against the toxic effects on yeast cells. On the genes of 5,117 open reading frames (ORFs), as valid spots in a microarray, 997 were up-regulated, 1,259 were down-regulated and 2,861 remained unchanged. A detailed analysis of the microarray revealed the genes that were dynamically correlated to the function of the subcellular localization, energy/metabolism, various stress responses/cell homeostasis and detoxification. Significantly, the toxicities, caused by reactive oxygen species (ROS), metals and the other xenobiotics, were indicated in burned ash. Also, the possibility of mutagenicity of the burned ash was suggested on the basis of the DNA repair related genes.

Application of artificial neural network modelling to identify severely ill patients whose aminoglycoside concentrations are likely to fall below therapeutic concentrations.

OBJECTIVE: Identification of ICU patients whose concentrations are likely to fall below therapeutic concentrations using artificial neural network (ANN) modelling and individual patient physiologic data. METHOD: Data on indicators of disease severity and some physiologic data were collected from 89 ICU patients who received arbekacin (ABK) and 61 who received amikacin (AMK). Three-layer ANN modelling and multivariate logistic regression analysis were used to predict the plasma concentrations of the aminoglycosides (ABK and AMK) in the severely ill patients. RESULTS: Predictive performance analysis showed that the sensitivity and specificity of ANN modelling was superior to multivariate logistic regression analysis. For accurate modelling, a predictable range should be inferred from the data structure before the analysis. Restriction of the predictable region, based on the data structure, increased predictive performance. CONCLUSION: ANN analysis was superior to multivariate logistic regression analysis in predicting which patients would have plasma concentrations lower than the minimum therapeutic concentration. To improve predictive performance, the predictable range should be inferred from the data structure before prediction. When applying ANN modelling in clinical settings, the predictive performance and predictable region should be investigated in detail to avoid the risk of harm to severely ill patients.

Massive cell death of cerebellar granule neurons accompanied with caspase-3-like protease activation and subsequent motor discoordination after intracerebroventricular injection of vincristine in mice.

Vincristine, a microtubule-depolymerizing agent, is known to induce neuronal cell damage. Biochemical, histological and behavioral alterations were investigated after intracerebroventricular injection of vincristine in mice. Intracerebroventricular injection of vincristine caused caspase-3-like protease activation followed by nucleosomal release in the cerebellum. Histological examinations showed that vincristine-induced damage was relatively specific to granule cells in the cerebellum, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells were observed among these cells. Chromatin condensation, one of the criteria for apoptosis, was seen on electron microscopy. Behavioral changes, namely head movements, pivoting and backward walking, were observed in parallel with the increase of caspase-3-like protease activity and nucleosomal release. Furthermore, motor function tests (bulb balance test and rotating rod test) showed deficits of motor coordination ability. These observations suggest that intracerebroventricular vincristine causes massive apoptosis of cerebellar granule cells accompanied with caspase-3-like protease activation, leading to motor dysfunction, in this model. These vincristine-treated mice should be a useful in vivo model for examination of neuronal apoptosis, which might be involved in a variety of neurodegenerative diseases.

Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium.

DNA microarray technology enables genome-wide detection of cell response at the transcriptional level. We are planning to make bioassay systems that can detect environmental chemicals to screen for potential bioreactive agents. To develop a DNA microarray for our purposes, the changes in gene expression underlying the yeast stress response to cadmium were analyzed by a microarray of total mRNA. Cadmium is a potent cell poison known to cause oxidative stress by changing intracellular glutathione levels. We report here that not only the glutathione synthesis gene (GSH1) but also almost all transcripts of the enzymes involved in the sulfur amino acid metabolism, especially MET14 and MET17, were greatly induced after exposure to cadmium. While several common stress-responsive genes, such as HSP26, GRE1, HSP12, and DDR48, were up-regulated more than almost fourfold by cadmium, there were also 42 other genes up-regulated more than fourfold. Based on these results, we concluded that DNA microarrays are very useful instruments for creating new bioassay systems and finding genetic promoters of stress indicators.

Systemic and eosinophilic lesions in rats with spontaneous eosinophilia (mes rats).

The mes rat is from an inbred mutant colony of rats with spontaneous eosinophilia. In order to investigate the pathogenesis of the mes rat, the histopathology and hematology for 76 mes rats were examined at several weeks of age. Tissue eosinophilia developed at 8 weeks of age when the blood eosinophil was 500 cells per microliter or more. Subsequently, eosinophilia progressed with age, and splenic eosinophilopoiesis and erythropoiesis appeared simultaneously. Many inflammatory lesions were induced after 10 weeks of age when the blood eosinophils became 1,000 cells per microliter or more. Gastroenteritis and mesenteric lymphadenitis were seen in 44 of 47 (94%) and 31 of 47 (66%) rats, respectively, after 10 weeks of age. Aortitis that deteriorated with age was found in 19 of 39 (49%) rats after 12 weeks of age. Hepatic fibrosis was found in four rats that exhibited severe eosinophilia and anemia. These results are comparable to the features of a hypereosinophilic syndrome in humans and other animals.

Quantitative analysis of crystalline pharmaceuticals in powders and tablets by a pattern-fitting procedure using X-ray powder diffraction data.

A pattern-fitting procedure for quantitative analysis of crystalline pharmaceuticals in solid dosage forms using X-ray powder diffraction data is described. This method is based on a procedure for pattern-fitting in crystal structure refinement, and observed X-ray scattering intensities were fitted to analytical expressions including some fitting parameters, i.e. scale factor, peak positions, peak widths and degree of preferred orientation of the crystallites. All fitting parameters were optimized by the non-linear least-squares procedure. Then the weight fraction of each component was determined from the optimized scale factors. In the present study, well-crystallized binary systems, zinc oxide-zinc sulfide (ZnO-ZnS) and salicylic acid-benzoic acid (SA-BA), were used as the samples. In analysis of the ZnO-ZnS system, the weight fraction of ZnO or ZnS could be determined quantitatively in the range of 5-95% in the case of both powders and tablets. In analysis of the SA-BA systems, the weight fraction of SA or BA could be determined quantitatively in the range of 20-80% in the case of both powders and tablets. Quantitative analysis applying this pattern-fitting procedure showed better reproducibility than other X-ray methods based on the linear or integral intensities of particular diffraction peaks. Analysis using this pattern-fitting procedure also has the advantage that the preferred orientation of the crystallites in solid dosage forms can be also determined in the course of quantitative analysis.

Effects of ketamine on voltage-dependent calcium currents and membrane potentials in single bullfrog atrial cells.

PURPOSE: This study was undertaken to assess the effect of ketamine on L-type calcium channel current (I(Ca)) and membrane action potential in the bullfrog single atrial myocyte. METHODS: Bullfrog single atrial myocytes were prepared by enzymatic dispersion. Whole-cell voltage-clamp technique and current clamp technique were used to monitor I(Ca), membrane resting potential, and action potential. RESULTS: Ketamine (10(-5)-10(-3) M) showed dose-dependent inhibition of I(Ca) in a reversible manner. The 50% inhibitory concentration (IC(50)) of ketamine on I(Ca) was estimated to be 0.92 x 10(-5) M. Use-dependent block of I(Ca) was not observed. The resting membrane potential was depolarized at a high concentration (10(-4) M) of ketamine. Reduction of the plateau phase and prolonged duration of the action potential were observed in the presence of a high concentration of ketamine (10(-4) M). CONCLUSION: Ketamine has an inhibitory effect on I(Ca) in the bullfrog single atrial myocyte, and a high dose (10(-4) M) of ketamine prolonges the duration of the action potential. The mechanism of inhibition of I(Ca) seems to be a direct effect on the L-type calcium channel, not like an open channel blocker.

The relationship of social norms to use of services and caregiver burden in Japan.

PURPOSE: To develop an improved measure of "sekentei" (a social-psychological process that restricts behaviors that do not conform to social norms such as family caregiving) among family caregivers in Japan, and to describe the relationships among sekentei and caregiver's actual use of services, a reluctance to use services, and care burden. DESIGN: Descriptive correlational study. Family caregivers (N = 260) of impaired elders responded to a structured questionnaire. METHODS: Exploratory and confirmatory factor analyses were used to assess the construct validity of the sekentei scale for caregivers (SSC). With the SSC, the relationships among main variables were verified. FINDINGS: The SSC showed satisfactory reliability and validity. Sekentei was significantly correlated with care burden, but not to actual use or reluctance to use services. CONCLUSIONS: Sekentei is an important factor related to caregiver burden in Japan. Further research might include the extent to which sekentei is a factor in care burden in other cultures.

[Treatment of advanced Parkinson's disease].

After the advent of levodopa, treatment of Parkinson's disease has changed and the activity of daily life of patients has been remarkably improved; whereas, many patients experience various problems such as wearing-off, dyskinesia, dystonia, neuropsychiatric problems, and dysautonomia. Especially, wearing-off and dyskinesia emerge with the change of absorptional pattern of levodopa and could be solved by regulating the timing and the dose of it. Recently, some new drugs for Parkinson's disease have been available and we physician have a wide choice of them. It is important to make a careful choice of and manipulate of doses of drugs after understanding daily life of each patient enough.

Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone.

Pioglitazone, a thiazolidinedione (TZD) derivative, is an antidiabetic agent that improves hyperglycaemia and hyperlipidaemia in obese and diabetic animals via a reduction in hepatic and peripheral insulin resistance. The TZDs including pioglitazone have been identified as high affinity ligands for peroxisome proliferator-activated receptor (PPAR) gamma. The selectivity of pioglitazone for the human PPAR subtypes has not been reported, thus, we investigated the effect of pioglitazone on the human PPAR subtypes. Transient transactivation assay showed that pioglitazone is a selective hPPARgamma1 activator and a weak hPPARalpha activator. Binding assay indicated that the transactivation of hPPARgamma1 or hPPARalpha by pioglitazone is due to direct binding of pioglitazone to each subtype. Furthermore, pioglitazone significantly increased the apoA-I secretion from the human hepatoma cell line HepG2.