Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4-2 and by serum- and glucocorticoid-dependent kinase 1

Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na(+) channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na(+)-P(i)) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (I(P)). Exposure to 3 mM phosphate induces an I(P) in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2, significantly downregulates I(P), whereas the coinjection of (S422D)SGK1 markedly stimulates I(P) and even fully reverses the effect of Nedd4-2 on I(P). The effect of (S422D)SGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active (T308D,S473D)PKB, or inactive (K127N)SGK1. Moreover, (S422D)SGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.     

Simultaneous PET-MRI: a new approach for functional and morphological imaging

Noninvasive imaging at the molecular level is an emerging field in biomedical research. This paper introduces a new technology synergizing two leading imaging methodologies: positron emission tomography (PET) and magnetic resonance imaging (MRI). Although the value of PET lies in its high-sensitivity tracking of biomarkers in vivo, it lacks resolving morphology. MRI has lower sensitivity, but produces high soft-tissue contrast and provides spectroscopic information and functional MRI (fMRI). We have developed a three-dimensional animal PET scanner that is built into a 7-T MRI. Our evaluations show that both modalities preserve their functionality, even when operated isochronously. With this combined imaging system, we simultaneously acquired functional and morphological PET-MRI data from living mice. PET-MRI provides a powerful tool for studying biology and pathology in preclinical research and has great potential for clinical applications. Combining fMRI and spectroscopy with PET paves the way for a new perspective in molecular imaging.     

Pathogenesis of murine enterovirus myocarditis: virus dissemination and immune cell targets.

In order to identify organ and cellular targets of persistent enterovirus infection in vivo, immunocompetent mice (SWR/J, H-2q) were inoculated intraperitoneally with coxsackievirus B3 (CVB3). By use of in situ hybridization for the detection of enteroviral RNA, we show that CVB3 is capable of inducing a multiorgan disease. During acute infection, viral RNA was visualized at high levels in the heart muscle, pancreas, spleen, and lymph nodes and at comparably low levels in the central nervous system, thymus, lung, and liver. At later stages of the disease, the presence of enteroviral RNA was found to be restricted to the myocardium, spleen, and lymph nodes. To characterize infected lymphoid cells during the course of the disease, enteroviral RNA and cell-specific surface antigens were visualized simultaneously in situ in spleen tissue sections. In acute infection, the majority of infected spleen cells, which are located primarily at the periphery of lymph follicles, were found to express the CD45R/B220+ phenotype of pre-B and B cells. Whereas viral RNA was also detected in certain CD4+ helper T cells and Mac-1+ macrophages, no enteroviral genomes were identified in CD8+ cytotoxic/suppressor T cells. Later in disease, the localization of enteroviral RNA revealed a persistent type of infection of B cells within the germinal centers of secondary follicles. In addition, detection of the replicative viral minus-strand RNA intermediate provided evidence for virus replication in lymphoid cells of the spleen during the course of the disease. These data indicate that immune cells are important targets of CVB3 infection, providing a noncardiac reservoir for viral RNA during acute and persistent myocardial enterovirus infection.     

Human Trophoblast Cells Are Permissive to the Complete Replicative Cycle of Human Cytomegalovirus

Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.