Aim:  In developed countries including Japan, the transmission route of indigenous hepatitis E virus (HEV) infection is obscure. Accordingly, public health implications of indigenous HEV infection have not been well addressed. The aim of this study was to clarify the route of transmission of a small outbreak of acute hepatitis E and assess the public health implications of indigenous zoonotic HEV transmission. Methods:  Three patients with non-A, B and C acute hepatitis, two of whom presented in a critical condition, were assessed for HEV infection using polymerase chain reaction and their route of infection; the genome sequences of the infecting HEV were also analyzed. A phylogenetic tree based on the full, or near full, HEV RNA sequences were constructed by neighbor-joining method. Results:  All three patients ingested grilled pork meat and entrails at the same barbecue restaurant in Abashiri, Hokkaido, Japan. When comparing partial to entire, or nearly entire, nucleotide sequences of HEV detected in these patients, they were 99.9-100% identical to each other. These genotype 4 isolates had great resemblance to the genome sequences of the isolates from the mini-outbreak in 2004 in Kitami, a city adjacent to Abashiri. These Kitami/Abashiri strains were segregated into a single cluster on the phylogenetic tree of HEV genotype 4 indigenous to Japan. Conclusion:  Indigenous HEV transmission via a zoonotic food-borne route has been demonstrated in Kitami and Abashiri via pork meat and entrails contaminated with virulent HEV strains. Because a similar outbreak can recur in the future, infection sources and distribution routes should be clarified rapidly for public health.

Activation of mesangial cells (MCs), which is characterized by induction of smooth muscle α-actin (SMA) expression, contributes to a key event in various renal diseases; however, the mechanisms controlling MC differentiation are still largely undefined. Activated Smad1 induce SMA in a dose-dependent manner in MCs. As a direct regulating molecule for SMA, we identified and characterized Scleraxis (Scx) as a new phenotype modulator in advanced glycation end-product (AGE)-exposed MCs. Scx physically associated with E12 and bound the E-box in the promoter of SMA and negatively regulated the AGE-induced SMA expression. Scx induced expression and secretion of bone morhogenetic protein 4 (BMP4), thereby controlling the Smad1 activation in AGE-treated MCs. In diabetic mice, Scx was concomitantly expressed with SMA in the glomeruli. Inhibitor of differentiation 1 (Id1) was further induced by extended treatment with AGE, thereby dislodging Scx from the SMA promoter. These data suggest that Scx and Id1 are involved in the BMP4-Smad1-SMA signal transduction pathway besides TGFβ1-Smad1-SMA signaling pathway and modulate phenotypic changes in MCs in diabetic nephropathy.

We investigated the molecular mechanisms underlying phloxine B (PhB)-induced photocytotoxicity in human T lymphocytic leukemia Jurkat cells. In addition to apoptosis-related biochemical events, photo-irradiated PhB generated intracellular reactive oxygen species (ROS), induced phosphorylation of c-Jun-N-terminal kinase (JNK) in an oxidative stress-dependent manner and up-regulated the gene expression of interferon (IFN)-γ, an inducer of diverse apoptosis-related molecules in activated T cells. PhB-induced apoptosis was significantly inhibited by N-acetyl-l-cysteine, but not by catalase, indicating that ROS generation occurred intracellularly, and by SP600125 and AG490, specific inhibitors of JNK and IFN-γ signaling, respectively, confirming their roles in the apoptotic pathway. IFN-γ up-regulation was also inhibited by SP600125, indicating that it was downstream of JNK activation. These results suggest that PhB-induced apoptosis in Jurkat cells partially involves the intracellular oxidative stress-sensitive and T cell-specific IFN-γ pathway. These data present a novel insight into the mechanisms of photocytotoxicity induced by artificial food colorants in human T lymphocytic leukemia cells.

Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-α production in rat basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced analog, 5',6'-dimethyl-1,1':4'1″-terphenyl-2',3',4,4″-tetraol (2) with a comparable inhibitory activity (IC(50)=0.02nM) to that of 1. The synthesis involved double Suzuki-Miyaura coupling as a key step, and required only five steps from commercially available 3,4-dimethylphenol. For identification of the target molecule, fluorescent and biotinylated derivatives of 2 were prepared through a 'click' coupling process.

RNA interference (RNAi) is a potent and highly specific gene-silencing phenomenon that was first reported for the nematode Caenorhabditis elegans. It has been discovered that genes could be silenced by introducing double-stranded RNAs (dsRNAs) complementary to the messenger RNA sequences. Since then, RNAi has been shown as an evolutionarily well-conserved process that plays an important role in host defense and in regulation of gene expression. Much effort has been dedicated to the application of the short dsRNA species (short interfering RNAs; siRNAs) as therapeutic agents, as they were shown to be effective in mammalian cells. Recently, we altered the structure of a siRNA molecule and produced dumbbell-shaped nanocircular RNAs. RNA dumbbells were shown to be stabilized in serum compared with its siRNA counterpart, despite their natural RNA strand. It has also been found that RNA dumbbells containing a 23-bp stem and two 9-nt loops exhibit a prolonged RNAi effect in cultured mammalian cells. In this unit, we describe the synthesis of RNA dumbbells from the design, its enzymatic synthesis, and to the purification. Curr. Protoc. Nucleic Acid Chem. 48:16.4.1-16.4.11. © 2012 by John Wiley & Sons, Inc.

Pre-treatment with α-tocopherol (α-Toc) potentiated cytotoxicity induction by benzyl isothiocyanate (BITC). Biochemical events related to apoptosis, such as DNA ladder formation and caspase-3 activation, were also enhanced by α-Toc. These results suggest a significant role of the caspase-3 pathway in apoptosis induction regulated by α-Toc in combination.

In the present study, we examined the toxicity of benzyl ITC (BITC) and its urinary mercapturic acid metabolite (BITC-NAC), using a normal renal proximal tubular cell line, pig LLC-PK1. BITC increased cell death with an IC(50) value of about 7 μM, whereas the cytotoxic effect of BITC-NAC was five times weaker than that of BITC. We observed a significant necrosis of the compounds on LLC-PK1 cells with oxidative stress. In the presence of 5 mM glutathione (GSH), comparable to physiological levels, the cytotoxicity of BITC-NAC as well as BITC was significantly reduced. Furthermore, the increase in intracellular GSH levels by pretreatment with NAC before the BITC treatment resulted in inhibition of the BITC-induced necrotic events as well as intracellular oxidative stress. These results suggest that GSH is a determinant of cellular resistance against the BITC-mediated and oxidative stress-dependent cytotoxicity in renal proximal tubular cells.

A better understanding of salt tolerance in plants might lead to the genetic engineering of crops that can grow in saline soils. Here we cloned and characterized plasma membrane and vacuolar Na(+)/H(+) antiporters of a monocotyledonous alkaline-tolerant halophyte, Puccinellia tenuiflora. The predicted amino acid sequence of the transporters were very similar to those of orthologs in monocotyledonous crops. Expression analysis showed that (1) NHA was more strongly induced by NaCl in the roots of P. tenuiflora while in rice it was rather induced in the shoots, suggesting that the role of NHA in salt excretion from the roots partly accounts for the difference in the tolerance of the two species, and that (2) NHXs were specifically induced by NaHCO(3) but not by NaCl in the roots of both species, suggesting that vacuolar-type Na(+)/H(+) antiporters play roles in pH regulation under alkaline salt conditions. Overexpression of the antiporters resulted in increased tolerance of shoots to NaCl and roots to NaHCO(3). Overexpression lines exhibited a lower Na(+) content and a higher K(+) content in shoots under NaCl treatments, leading to a higher K(+)/Na(+) ratio.

During the 2009-2010 season, a significant numerical increase of genotype GII.2 norovirus (NoV)-associated outbreaks was observed in Osaka City, Japan. The most common genotype in that season was GII.2 (44.6%), followed by GII.4 (39.2%). Mostly, GII.2 strains were associated with outbreaks in children and with person-to-person contact. The National Infectious Disease Surveillance Center reported that GII.2 NoV infections were widespread in Japan in that season. Comparative phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) and capsid sequences revealed that this GII.2 epidemic resulted from two genetic strains. The first, GII.2p2 strains, had an identical genotype in the RdRp and capsid genes. GII.2p2 strains in the 2009-2010 season were a different genetic cluster from the strains of spring 2004, the previous epidemic of GII.2 NoV, but showed no unique amino acid change. The second, GII.2 chimera virus (GII.2p16), had GII.16 RdRp and GII.2 capsid genotypes, suggesting prior recombination at the junction of ORF1 and ORF2. GII.2p16 strains had four significant amino acid changes in the P2 subdomain, suggesting antigenic changes. Before the 2009-2010 season, GII.2 chimera viruses had been observed only sporadically. This spreading of GII.2p16 strains in the 2009-2010 season might be the first epidemic of GII.2 chimera virus. This study revealed that the NoV epidemic in the 2009-2010 season differed considerably from the prior season, when GII.4 was predominant. Furthermore, GII.2 strains persisted in human populations by drastic recombination and gradual accumulation of mutations, indicating a prevalent pattern of non-GII.4 genotypes with genetic evolution. J. Med. Virol. 84:517-525, 2012. © 2011 Wiley Periodicals, Inc.

Scolecobasidium, generally found in outdoor samples, were isolated from detergent-rich indoor environments. The isolates from bathrooms and washing machines, because of their exposure to detergents, might be genetically and biologically distinct from outdoor isolates. In this study, 11 Scolecobasidium isolates from detergent-rich indoor environments were examined to find the genetic and biological differences between the indoor and outdoor isolates. One isolate from a wall of a soap factory, showing similar conidia morphology with S. constricta, was phylogenetically distinct from the other Scolecobasidium spp. The 10 isolates from washing machines and bathrooms were identified as S. humicola, but these were classified into 2 groups that differed from the reference strain of S. humicola from leaves. All 11 isolates and the 4 reference strains of S. constricta and S. humicola grew on the medium containing sodium oleate and polyoxyethylene-(9)-lauryl ether, but the reference strains of the other Scolecobasidium spp. grew only on the medium containing sodium oleate. The results showed that S. humicola and S. constricta could utilize both surfactants generally included in soaps or synthetic detergents as nutrients. A further implication is that the genetic variation found in the S. humicola isolates from detergent-rich indoor environments can occur as a result of adaptation to such an environment.