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Selecting the best quantitative PCR assay is essential to detect human norovirus genome effectively from clinical and environmental samples because no cell lines have been developed to propagate this virus. The real-time PCR methods for noroviruses GI (4 assays) and GII (3 assays) were evaluated using wastewater (n = 70) and norovirus-positive stool (n = 77) samples collected in Japan between 2012 and 2013. Standard quantitative PCR assays recommended by the U.S. Environmental Protection Agency, International Organization for Standardization, and Ministry of Health, Labour and Welfare, Japan, together with recently reported assays were included. Significant differences in positive rates and quantification cycles were observed by non-parametric analysis. The present study identifies the best assay for norovirus GI and GII to amplify norovirus genomes efficiently. [ABSTRACT FROM AUTHOR]

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [Ptdlns(3,5)P2] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of Ptdlns(3,5)P2 in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P2, is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that Ptdlns(3,5)P2 metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

The present study analyses changes in nitrogen compounds, amino acid composition, and glutamate metabolism in the resurrection plant Sporobolus stapfianus during dehydration stress. Results showed that older leaves (OL) were desiccation-sensitive whereas younger leaves (YL) were desiccation-tolerant. OL lost their soluble protein more rapidly, and to a larger extent than YL. Enzymes of primary nitrogen assimilation were affected by desiccation and the decrease in the glutamine synthetase (GS, EC 6.3.1.2) and ferredoxin-dependent GOGAT (Fd-GOGAT, EC 1.4.7.1) activities was higher in OL than in YL, thus suggesting higher sensibility to dehydration. Moreover, YL showed higher total GS enzyme activity at the end of the dehydration stress and was shown to maintain high chloroplastic GS protein content during the entire stress period. Free amino acid content increased in both YL and OL between 88% and 6% relative water content. Interestingly, OL and YL did not accumulate the same amino acids. OL accumulated large amounts of proline and γ-aminobutyrate whereas YL preferentially accumulated asparagine and arginine. It is concluded (i) that modifications in the nitrogen and amino acid metabolism during dehydration stress were different depending on leaf development and (ii) that proline and γ-aminobutyrate accumulation in S. stapfianus leaves were not essential for the acquisition of desiccation tolerance. On the contrary, the accumulation of large amounts of asparagine and arginine in the YL during dehydration could be important and serve as essential nitrogen and carbon reservoirs useful during rehydration. In this context, the role of GS for asparagine accumulation in YL is discussed. [ABSTRACT FROM PUBLISHER]

We have characterized the biochemical properties of the testis and brain-specific 105-kDa protein which is cross-reacted with an anti-bovine HSP90 antibody. The protein was induced in germ cells by heat stress, resulting in a protein which is one of the heat shock proteins [Kumagai, J., Fukuda, J., Kodama, H., Murata, M., Kawamura, K., Itoh, H. & Tanaka, T. (2000) Eur. J. Biochem. 267 , 3073–3078]. In the present study, we characterized the biochemical properties of the protein. The 105-kDa protein inhibited the aggregation of citrate synthase as a molecular chaperone in vitro . ATP/MgCl2 has a slight influence of the suppression of the citrate synthase aggregation by the 105-kDa protein. The protein possessed chaperone activity. The protein was able to bind to ATP–Sepharose like the other molecular chaperone HSP70. A partial amino-acid sequence (24 amino-acid residues) of the protein was determined and coincided with those of the mouse testis- and brain-specific APG-1 and osmotic stress protein 94 (OSP94). The 105-kDa protein was detected only in the medulla of the rat kidney sections similar to OSP94 upon immunoblotting. The purified 105-kDa protein was cross-reacted with an antibody against APG-1. These results suggested that APG-1 and OSP94 are both identical to the 105-kDa protein. There were highly homologous regions between the 105-kDa protein/APG-1/OSP94 and HSP90. The region of HSP90 was also an immunoreactive site. An anti-bovine HSP90 antibody may cross-react with the 105-kDa protein similar to HSP90 in the rat testis and brain. We have investigated the localization and developmental induction of the protein in the rat brain. In the immunohistochemical analysis, the protein was mainly detected in the cytoplasm of the nerve and glial cells of the rat brain. Although the 105-kDa protein was localized in all rat brain segments, the expression pattern was fast in the cerebral cortex and hippocampus and slow in the cerebellum. [ABSTRACT FROM AUTHOR]

Spinach leaf ferredoxin and ferredoxin : NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin- NADP-reductase - Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases. [ABSTRACT FROM AUTHOR]

Incubation of wild-type ferredoxin (Fd) with Chlamydomonas reinhardii crude extract in the presence of a carboxyl activator resulted in the formation of a unique 1:1 covalent complex with nitrite reductase. However, glutamate synthase was able to form two covalent complexes of Fd: GOGAT with 1:1 and 2:1 stoichiometries. These complexes were functional only when reduced methyl viologen was used as electron donor. Kinetic studies of complex formation suggested the presence of two Fd-binding domains with similar affinity for Fd in the glutamate synthase molecule. Using site-directed mutagenesis with recombinant Fd. we have shown that Fd-Glu91 is directly involved in Fd interaction with glutamate synthase and nitrite reductase. Moreover, a negative core of residues in the α1 helix of Fd was also critical in binding the enzymes. These data highlight the analogy in the Fd-binding sites of nitrite reductase and glutamate synthase, which may compete for the electrons coming from the photosynthetic chain. [ABSTRACT FROM AUTHOR]

Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. Here we show that insulin stimulates the phosphatase activity of Slingshot-IL (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increased in PTEN (phosphatase and tensin homolog deleted in chromosome 10)-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt (a downstream effector of PI3K), together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated coillin, accumulated in membrane protrusions in insulinstimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSHIL expression by RNA interference abolished insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSHIL is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin. [ABSTRACT FROM AUTHOR]