Distinct genotype and antigenicity among genogroup II sapoviruses

SaV, a pathogen of acute gastroenteritis, is divided into five genogroups, GI to GV. However, the relation between SaV antigenicity and genetic clusters is not fully understood. We have recently identified two GII SaV strains, Mc10 and C12, which are grouped into the same cluster based on the polymerase but are grouped into distinct clusters based on the capsid. To evaluate the difference in antigenicity between these two strains, VLP were expressed in mammalian cells. An antigen ELISA demonstrated for the first time that strains in the same GII SaV genogroup, but within different clusters, have distinct antigenicities.     

Calpain 6 is involved in microtubule stabilization and cytoskeletal organization

The calpains are a family of Ca(2+)-dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated alpha-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated alpha-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization.     

Norovirus Binding to Intestinal Epithelial Cells Is Independent of Histo-Blood Group Antigens

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le^b-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.     

Hepatic overexpression of a dominant negative form of raptor enhances Akt phosphorylation and restores insulin sensitivity in K/KAy mice

Several serine/threonine kinases reportedly phosphorylate serine residues of IRS-1 and thereby induce insulin resistance. In this study, to investigate the effect of mTOR/raptor on insulin signaling and metabolism in K/KAy mice with genetic obesity-associated insulin resistance, a dominant negative raptor, COOH-terminally deleted raptor (raptor-DeltaC(T)), was overexpressed in the liver via injection of its adenovirus into the circulation. Hepatic raptor-DeltaC(T) expression levels were 1.5- to 4-fold that of endogenously expressed raptor. Glucose tolerance in raptor-DeltaC(T)-overexpressing mice improved significantly compared with that of LacZ-overexpressing mice. Insulin-induced activation of p70S6 kinase (p70(S6k)) was significantly suppressed in the livers of raptor-DeltaC(T) overexpressing mice. In addition, insulin-induced IRS-1, Ser(307), and Ser(636/639) phosphorylations were significantly suppressed in the raptor-DeltaC(T)-overexpressing liver, whereas tyrosine phosphorylation of IRS-1 was increased. PI 3-kinase activation in response to insulin stimulation was increased approximately twofold, and Akt phosphorylation was clearly enhanced under both basal and insulin-stimulated conditions in the livers of raptor-DeltaC(T) mice. Thus, our data indicate that suppression of the mTOR/p70(S6k) pathway leads to improved glucose tolerance in K/KAy mice. These observations may contribute to the development of novel antidiabetic agents.