Uralsaponins M-Y, antiviral triterpenoid saponins from the roots of Glycyrrhiza uralensis.

Thirteen new oleanane-type triterpenoid saponins, uralsaponins M-Y (1-13), and 15 known analogues (14-28) were isolated from the roots of Glycyrrhiza uralensis Fisch. The structures of 1-13 were identified on the basis of extensive NMR and MS data analyses. The sugar residues were identified by gas chromatography and ion chromatography coupled with pulsed amperometric detection after hydrolysis. Saponins containing a galacturonic acid (1-3) or xylose (5) residue are reported from Glycyrrhiza species for the first time. Compounds 1, 7, 8, and 24 exhibited good inhibitory activities against the influenza virus A/WSN/33 (H1N1) in MDCK cells with IC50 values of 48.0, 42.7, 39.6, and 49.1 µM, respectively, versus 45.6 µM of the positive control oseltamivir phosphate. In addition, compounds 24 and 28 showed anti-HIV activities with IC50 values of 29.5 and 41.7 µM, respectively.

Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.

Organotypic slice culture preserves the morphological and physiological features of the hippocampus of live animals for a certain time. The hippocampus is one of exceptional regions where neurons are generated intrinsically and spontaneously throughout postnatal life. We investigated the possibility that neurons are generated continuously at the dentate granule cell layer (GCL) in slice culture of the rat hippocampus. Using 5-bromodeoxyuridine (BrdU) labelling and retrovirus vector transduction methods, the phenotypes of the newly generated cells were identified immunohistochemically. At 4 weeks after BrdU exposure, BrdU-labelled cells were found in the GCL and were immunoreactive with a neuronal marker, anti-NeuN. There were fibrils immunoreactive with anti-glial fibrillary acidic protein (GFAP), an astrocyte marker, in the layer covering the GCL and occasionally encapsulated BrdU-labelled nuclei. When the newly divided cells were marked with the enhanced green fluorescent protein (EGFP) using a retrovirus vector, these cells had proliferative abilities throughout the following 4-week cultivation period. Four weeks after the inoculation, the EGFP-expressing cells consisted of various phenotypes of both early and late stages of differentiation; some were NeuN-positive cells with appearances of neurons in the GCL and some were immunoreactive with anti-Tuj1, a marker of immature neurons. Some EGFP-expressing cells were immunoreactive with anti-GFAP or anti-nestin, a marker of neural progenitors. The present study suggests that slice cultures intrinsically retain spontaneous neurogenic abilities for their cultivation period. The combination of slice culture and retrovirus transduction methods enable the newly divided cells to be followed up for a long period.

Activation of murine cytomegalovirus immediate-early promoter in mouse brain after transplantation of the neural stem cells.

Cytomegalovirus (CMV) is the most significant infectious cause of congenital infection and fatal diseases in immunocompromised patients. We have previously described a transgenic mouse model of the murine CMV (MCMV) immediate-early (IE) gene promoter fused with the lacZ reporter gene (MCMV-IE-pro1) for the analysis of spatiotemporal changes of the promoter activity during brain development. Since expression of the IE genes play a pivotal role in latency and reactivation, we transplanted the transgene-expressing neural stem cells (NSCs) into neonatal mouse brains after labeling with bromodeoxyuridine (BrdU). The activation of MCMV-IE pro1 was detected in the subventricular zone (SVZ) soon after transplantation, and the number of MCMV IE pro1-activated cells was decreased as the development proceeded. Cells that were MCMV IE pro1-activated and glial fibrillary acidic protein positive, but not stained with BrdU, were found in the cortex 4 weeks after transplantation, while BrdU-positive but not MCMV IE pro1-activated cells still existed in the SVZ. MCMV IE promoter activity tended to be easily detected in the cortex after allogenic transplantation in BALB/c mouse. These results suggest that the SVZ is the most susceptible site for activation of the MCMV IE promoter in neonatal mice in the early period after transplantation and that the cerebral cortex is also susceptible to the activation in the late period after transplantation. It may be important to avoid the use of NSCs latently infected with CMV as donor cells.

Neuron-specific activation of murine cytomegalovirus early gene e1 promoter in transgenic mice.

The brain is the main target in congenital cytomegalovirus (CMV) infection and immunocompromised patients. No definite evidence that a CMV has special affinity for the central nervous system (CNS) has been published. Here, we generated transgenic mice with an e1 promoter/enhancer region connected to the reporter gene lacZ. Surprisingly, expression of the transgene was completely restricted to the CNS in all lines of transgenic mice. The transgene was expressed in subpopulation of neurons in the cerebral cortex, hippocampus, diencephalon, brainstem, cerebellum, and spinal cord in all of the lines. Non-neuronal cells in the CNS were negative for transgene expression. Activation of the transgene was first observed in neurons of mesencephalon in late gestation, and then the number of positive neurons increased in various parts of the brain as development proceeded. Upon infection of the transgenic mouse brains with MCMV, the location of the activated neurons became more extensive, and the number of such neurons increased. These results suggest that there are host factor(s) that directly activate the MCMV early gene promoter in neurons. This neuron-specific activation may be associated with persistent infection in the brain and may be responsible for the neuronal dysfunction and neuronal cell loss caused by CMV infection.