Predominance of PCR-ribotypes, 018 (smz) and 369 (trf) of Clostridium difficile in Japan: a potential relationship with other global circulating strains?

Global spread and evolutionary links of an epidemic Clostridium difficile strain (PCR-ribotype 027) have been noted in recent decades. However, in Japan, no outbreaks caused by type 027 have been reported to date. A total of 120 C. difficile isolates from patients at 15 hospitals during non-outbreak seasons between 2011 and 2013 as well as 18 and 21 isolates collected from two hospitals in 2010 and 2009, respectively, in outbreak periods in Japan, were examined. Among these 120 isolates, Japan-ribotypes smz and ysmz (subtype variant of smz) were the most predominant (39.2 %) followed by Japan-ribotype trf (15.8 %). Types smz/ysmz and trf were also concurrently predominant at two hospitals in the outbreak settings. Out of the five binary toxin-positive isolates observed, only one was PCR-ribotype 027 and another PCR-ribotype 078. Type smz was later found to correspond to PCR-ribotype 018. High rates of resistance against gatifloxacin, moxifloxacin, erythromycin and clindamycin were observed in the PCR-ribotype 018 isolates. Interestingly, all trf isolates were toxin A-negative, toxin B-positive, but they did not correspond to PCR-ribotype 017, thus being assigned a new ribotype (PCR-ribotype 369). In conclusion, PCR-ribotypes 018 (smz) and 369 (trf) were identified as major circulating strains in both outbreak and non-outbreak settings in Japan. Given their epidemiological relevance, molecular investigations are warranted to clarify potential evolutionary links with related strains found elsewhere, such as PCR-ribotypes 018 and 017 from Europe and North America.

Three-dimensional rational approach to the discovery of potent substituted cyclopropyl urea soluble epoxide hydrolase inhibitors.

We have previously reported a series of cyclopropyl urea derivatives as potent orally available soluble epoxide hydrolase (sEH) inhibitors. Here, we designed and synthesized three substituted cyclopropane derivatives that occupy all available pockets of sEH catalytic domain. Compound 14 with a diphenyl substituted cyclopropyl moiety showed good sEH inhibitory activity. Co-crystal structure of this compound and human sEH hydrolase catalytic domain revealed enzyme pockets occupied by the phenoxypiperidine part and the diphenyl cyclopropyl moiety. Furthermore, investigation of the phenoxypiperidine part of compound 14 resulted in the discovery of compound 19, which showed potent sEH inhibitory activity (sub-nM sEH IC50 values).

Involvement of diacylglycerol produced by phospholipase D activation in Aß-induced reduction of sAPPα secretion in SH-SY5Y neuroblastoma cells.

We previously reported that the thiol proteinase inhibitor, E-64-d, ameliorated amyloid ß (Aß)-induced reduction of soluble amyloid precursor protein α (sAPPα) secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells. In the present study, we showed that Aß (1-42) peptide enhanced diacylglycerol (DAG) production by phospholipase D (PLD) activation in these cells. We subsequently examined whether PLD was involved in Aß-induced reduction of sAPPα secretion and showed that 2 µM CAY10593, which selectively inhibits PLD2, ameliorated reduction of sAPPα secretion, whereas 50 nM CAY10593, which selectively inhibits PLD1, did not. Moreover, 50 µM propranolol, a phosphatidic acid phosphohydrolase inhibitor, also ameliorated Aß-induced reduction of sAPPα secretion, suggesting that DAG may be responsible for Aß-induced reduction of sAPPα. We subsequently examined whether DAG affects sAPPα secretion and showed that a DAG analog reduced sAPPα secretion in SH-SY5Y cells. In addition, DAG enhanced ceramide production by stimulating neutral sphingomyelinase (N-SMase) activity. We previously demonstrated that Aß stimulates N-SMase activity in SH-SY5Y cells. Here, we showed that inhibition of PLD2 by 2 µM CAY10593 suppressed Aß-induced N-SMase activation. Taken together, the results suggest that DAG produced through the PLD pathway is involved in Aß-induced reduction of sAPPα secretion in SH-SY5Y cells.

Structure-based optimization of cyclopropyl urea derivatives as potent soluble epoxide hydrolase inhibitors for potential decrease of renal injury without hypotensive action.

Epoxyeicosatrienoic acids (EETs) are known to have beneficial pharmacological effects on various cardiovascular events. However, EETs are biologically metabolized by soluble epoxide hydrolase (sEH) to less active metabolites. In our search for potent sEH inhibitors, we optimized a series of cyclopropyl urea derivatives and identified compound 38 as a potent sEH inhibitor with minimal CYP inhibition and good oral absorption in rats. Administration of 38 to DOCA-salt rats suppressed urinary albumin and MCP-1 excretion without affecting systolic blood pressure.

The thiol proteinase inhibitor E-64-d ameliorates amyloid-ß-induced reduction of sAPPα secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells.

In Alzheimer's disease (AD), enhancing α-secretase processing of amyloid precursor protein (APP) is an important pathway to decrease neurotoxic amyloid ß (Aß) secretion. The α-secretase is reported to be regulated by protein kinase C (PKC) and various endogenous proteins or cell surface receptors. In this report, we first examined whether Aß reduces α-secretase activity, and showed that Aß peptide 1-40 (0.001 and 0.01 µM) reduced the secretion of soluble amyloid precursor protein α (sAPPα) in carbachol-stimulated SH-SY5Y neuroblastoma cells. E-64-d (3 µM), which is a potent calpain inhibitor that prevents PKC degradation, ameliorated the Aß-induced reduction of sAPPα secretion. In addition, we observed that Aß significantly enhanced ceramide production by activating neutral sphingomyelinase. The cell-permeable ceramide analog, C2-ceramide (1 µg/mL), also reduced sAPPα secretion, and in addition, E-64-d eliminated the observed decrease of sAPPα secretion. C2-ceramide induced down-regulation of PKC-α, -ß1, and -ß2 isozymes in SH-SY5Y cells. These findings suggest that ceramide may play an important role in sAPPα processing by modulating PKC activity.

Subcutaneous abscess formation in the upper extremity caused by toxigenic Corynebacterium ulcerans.

Corynebacterium ulcerans is attracting attention as an emerging zoonosis that causes lymphadenitis, dermatitis and respiratory infections. We report here what appears to be the first case of subcutaneous abscess formation in the upper extremity due to toxigenic C. ulcerans in Japan. Awareness of the fact that C. ulcerans can cause a subcutaneous, elastic-hard, less-mobile mass with heat, redness and pain in the extremities is important for differential diagnosis.

Asymmetric synthesis of the carbon-14-labeled selective glucocorticoid receptor modulator using cinchona alkaloid catalyzed addition of 6-bromoindole to ethyl trifluoropyruvate.

We describe in this study the asymmetric synthesis of radioisotope (RI)-labeled selective glucocorticoid receptor modulator. This synthesis is based on optimization of the cinchona alkaloid catalyzed addition of 6-bromoindole to ethyl trifluoropyruvate and Negishi coupling of zinc cyanide to the 6-bromoindole moiety. [¹4C] Labeled (-)-{4-[(1-{2-[6-cyano-1-(cyclohexylmethyl)-1H-indol-3-yl]-3,3,3-trifluoro-2-hydroxypropyl}piperidin-4-yl)oxy]-3-methoxyphenyl}acetic acid (-)-1 was synthesized successfully with high enantioselectivity (>99% ee) and sufficient radiochemical purity.

Removal characteristics of wipe devices under various conditions.

Understanding the removal factor for specific conditions is essential to estimate removable surface contamination levels by wiping. The removal characteristics of dry foamed polystyrene pads and filter paper applied to polyvinyl chloride flooring are investigated using C-thymidine under various conditions (i.e., weight of contaminants, wax coating, temperature and humidity). Eight wipes were performed per flooring piece with a uniformly deposited contaminant to estimate the total removable surface contamination. The wipe devices were pressed against the surface by 2 kg-force with fingertips. The first wipe ratio (the ratio of the activity removed by the first wipe to the activity removed by eight wipes) from flooring on which was deposited 500 microg mL of C-thymidine solution varies between 24% and 71% for polystyrene pads and between 33% and 83% for filter papers, depending on the atmospheric humidity and temperature. For deposition concentrations of 1 microg mL, the mean first wipe ratio using polystyrene pads, which are almost constant vs. humidity and temperature, are 68% and 24% for waxed and unwaxed surfaces, respectively. Under the same conditions, the mean first wipe ratio for filter paper varied with both the surface and the environmental conditions. The total recovery (the ratio of the total activity removed by eight wipes to the total deposited activity) for deposition concentrations of 500 microg mL tended to increase with humidity for both wiping devices. The total recovery for deposition concentrations of 1 microg mL is generally low. The first wipe ratio in the lowest case with foamed polystyrene pads in this study was 19%. In that case, the total removable activity is speculated to increase from the sum of eight wipes, if additional wipes are performed. Therefore, the theoretical value of 0.1 recommended by ISO for the removal factor is appropriate when wiping polyvinyl chloride flooring using foamed polystyrene pads.

Investigation of monitoring for internal exposure by urine bioassay in a biomedical research facility.

A simple monitoring programme by urine bioassay was carried out to assess internal doses for workers in a biomedical research facility. Urine samples were measured without chemical treatment using a liquid scintillation counter or sodium iodide (NaI (Tl)) scintillation detector. The detection limits for the committed effective doses were below 1 mSv for 125I and 131I and below 1 x 10(-1) mSv for 3H, 14C, 32P, 35S and 51Cr, in the case that samples were collected within 3 d after handling. All of the urinary concentrations were below the detection limit, suggesting that no significant intake was detected during the investigation. The present results suggest that personal monitoring, such as the urine bioassay, is not necessary in many cases as a routine monitoring for workers engaged in tracer experiments using the above nuclides.

Expression pattern of FOS in orexin neurons during sleep induced by an adenosine A2A receptor agonist.

The present study examined the expression pattern of FOS in the hypothalamic peptide neurons during the sleep-dominant state induced by an adenosine A2A receptor agonist. The control rats, those that received the microdialysis-perfusion of their ventral striatum with artificial cerebrospinal fluid in the dark-active phase, spent 24% of the 90-min period prior to sacrifice in non-rapid eye movement (non-REM) sleep and 2.3% of that in REM sleep. These rats exhibited FOS, a transcription factor, in 21% of their orexin neurons and in 1.0% of their melanin-concentrating hormone (MCH) neurons in the perifornical/lateral hypothalamic areas. However, the rats perfused with 50 microM CGS21680, an adenosine A2A receptor agonist, spent 60% of the 90-min period prior to sacrifice in non-REM sleep and 11% of that in REM sleep. These rats exhibited FOS in 1.7% of their orexin neurons and FOS in 0.5% of their MCH neurons. When the sleep-dominant state was disturbed by mild stimulation and the rats were kept in the sleepy state by treatment with a sleep-inducing dose of CGS21680, the rats exhibited FOS in 13.3% of their orexin neurons, which percentage was about half of that for the control rats. These results suggest that the sleep-promoting process induced by this adenosine A2A receptor agonist was associated with a decline in the activity of orexin neurons. MCH neurons are not likely to change their activities during this sleep-promoting process.

Genetic marking of hematopoietic stem and endothelial cells: identification of the Tmtsp gene encoding a novel cell surface protein with the thrombospondin-1 domain.

Using an in silico database search, we identified a novel gene encoding a cell surface molecule with a thrombospondin domain, and designated the gene as transmembrane molecule with thrombospondin module (Tmtsp). Expression profiling of Tmtsp using specific monoclonal antibodies and Venus, a variant of yellow fluorescent protein knock-in mice in the Tmtsp locus, demonstrated its specific expression in hematopoietic and endothelial cells. In lymphohematopoietic cells, Tmtsp was predominantly expressed in hematopoietic stem and progenitor cells, and the level of expression gradually declined as the cells differentiated. Venus expression faithfully traced the expression of Tmtsp, and the level of Venus expression correlated well to the in vitro hematopoietic activity as well as the in vivo bone marrow repopulating capacity. Notably, Venus expression marked the development of definitive hematopoiesis in both the extraembryonic yolk sac and the intraembryonic aorta-gonad-mesonephros (AGM) region and, in combination with CD41, strikingly promoted the enrichment of developing progenitors in the CD41(+)Venus(high) fraction at embryonic day 10.5 (E10.5). In this context, Tmtsp is a novel marker gene for primitive hematopoietic cells and endothelial cells, and Tmtsp(Venus/)(+) mice would serve as a valuable mouse model for the analysis of both embryonic and adult hematopoiesis, as well as for vascular biology.

Establishment and issues of new educational system by nutrition teachers.

The Japanese school lunch system, which has over 100 y of history, has gained world attention for its well-developed and steady system, healthy Japanese-style menus including rice, and educational programs utilizing the school lunch system. Meanwhile, risk factors of health among students have been continuously worsening for the past dozen years or so. Therefore, Japan had hastened to develop better educational programs utilizing the school lunch and nutritional teachers. Under such circumstances, the Japanese Diet passed a bill to amend the School Education Law (hereinafter called the Amendment) which includes the establishment of an educational system by nutrition teachers in May, 2004. This system will be enforced in April, 2005. This system by nutrition teachers in Japan is well organized in both the legal and administrative senses and is attracting domestic and international attention as an example of an educational measure to improve the dietary life of students. The reason for this article in English is the additional intention of enlightening other countries by showing the political promotion problems of educational administration agencies, educational contents of nutrition teachers and some issues of the new educational system by nutrition teachers.

A case study of the estimation of occupational internal dose using urinary excretion data obtained in a biomedical research facility.

This paper presents a fundamental investigation of the screening of intakes and the estimation of internal doses for workers at a biomedical research facility from their urinary excretion data. Urine samples from the workers were collected immediately before and several days after handling radioactive materials, and 5-mL portions were measured directly with a sodium iodide scintillation detector or a liquid scintillation detector. Intakes of radionuclides and effective doses were estimated from the data using the Internal Dose Easy Calculation code. Blank levels of radioactivity and detection limits for this method were determined from the radioactivity contained in urine samples collected from volunteers working outside the controlled area (control samples). Most of the radioactivity in the control samples was attributed to K. Under the present experimental conditions, the detection limits for the committed effective doses were 7.5 x 10(-4) mSv for 125I and 6.9 x 10(-2) mSv for 35S in the case of samples collected 1 d after handling. Accordingly, it can be determined whether workers were exposed more than the recording level (1 mSv of committed effective dose) or not. No workers in the surveyed facility were found to suffer internal exposure beyond the recording levels, and their intakes were estimated to be below the detection limits in the 2-y investigation period. In an ordinary facility of biomedical research, the amount of unsealed radioactivity handled at one time is within 10 MBq in many cases, and the air ventilation and filtering systems work adequately. Under such circumstances, the present results suggest that individual monitoring, such as urine analysis, is omissible in many cases as a routine monitoring of internal exposure for workers.

Relationship between CDX2 gene methylation and dietary factors in gastric cancer patients.

Epigenetic gene silencing through DNA methylation is one of the important steps in the mechanism underlying tumorigenesis, including in the stomach. Past lifestyle factors of cancer patients, such as intake of vegetables, are very important in affecting gastric carcinogenesis. However, the relationship between DNA methylation and past dietary habits in cancer patients remains largely unknown. The CDX2 homeobox transcription factor plays a key role in intestinal development, but CDX2 is also expressed in most of the intestinal metaplasia and part of the carcinomas of the stomach. We analyzed the methylation status of the CDX2 5' CpG island in gastric cancer cell lines by methylation-specific PCR (MSP), and then CDX2 mRNA was found to be activated after 5-aza-2'-deoxycytidine treatment of the methylation-positive cells. We further examined the methylation status of CDX2 in primary gastric carcinomas by MSP and compared it with the past lifestyle of the patients, including dietary habits. Methylation of CDX2 was found in 20 (34.5%) of the 58 male patients and one (6.7%) of the 15 female patients. Since the methylation frequency was low in the female patients, the analysis was performed only on the male cases. CDX2 methylation was correlated with the decreased intake of green tea and cruciferous vegetables, and also with full or overeating habits. These findings are consistent with epidemiological observations on gastric cancer. We also analyzed the methylation status of p16/INK4a and hMLH1, but their frequencies were not associated with dietary factors or other lifestyle factors. Thus, diet could be an important factor determining the methylation status of genes such as CDX2 and the resultant aberrant expression of genes involved in carcinogenesis.

FOS expression in orexin neurons following muscimol perfusion of preoptic area.

Unilateral microdialysis-perfusion of the preoptic area with 50 microM muscimol decreased the sleep period of rats to less than 3% of the baseline value over a 90 min period before death (p = 0.018 by Wilcoxon signed-rank test). These rats showed the expression of FOS in 36% of the orexin neurons located in the perifornical/lateral hypothalamic areas on the side ipsilateral to the perfusion site, but in only 3% of the orexin neurons on the side contralateral to it (p = 0.018 by Wilcoxon signed-rank test). These results suggest that subpopulations of the preoptic neurons give an inhibitory tone to the activities of the orexin neurons in the perifornical/lateral hypothalamic areas.

Inhibition of rostral basal forebrain neurons promotes wakefulness and induces FOS in orexin neurons.

The present study examined whether the activities of the rostral basal forebrain neurons alter the activities of the orexin (also known as hypocretin) neurons in the tuberal part of the hypothalamus in rats. We performed microdialysis perfusion of the ventromedial portion of the rostral basal forebrain with the GABAA receptor agonist muscimol to inhibit focally the neuronal activities in the rostral basal forebrain. Then, we monitored sleep/wake behaviour and investigated the pattern of activities of orexin neurons by examining the expression of FOS as an indicator of cellular activation. Bilateral perfusion with muscimol (5, 15, and 50 micro m) produced a dose-dependent decrease in the amount of sleep. This perfusion with muscimol at 50 micro m produced FOS-like immunoreactivity in 37% of the orexin neurons located in the tuberal part of the hypothalamus, whereas the FOS-like immunoreactivity was sparse in orexin neurons of the sleeping control rats (P = 0.001 by Mann-Whitney U-test). Unilateral perfusion with muscimol (50 micro m) also suppressed sleep. In this case, FOS-like immunoreactivity was seen in 40% of the orexin neurons on the side ipsilateral to the perfusion site but only in 10% of orexin neurons on the contralateral side (P = 0.018 by Wilcoxon signed rank test). These functional data suggested that a sleep-generating element in the ventromedial part of the rostral basal forebrain provides an inhibitory influence on the activities of the orexin neurons in the tuberal part of the hypothalamus.